Methods for forming lipid bilayers on biochips

ABSTRACT

This disclosure provides a biochip comprising a plurality of wells. The biochip includes a membrane that is disposed in or adjacent to an individual well of the plurality of wells. The membrane comprises a nanopore, and the individual well comprises an electrode that detects a signal upon ionic flow through the pore in response to a species passing through or adjacent to the nanopore. The electrode can be a non-sacrificial electrode. A lipid bilayer can be formed over the plurality of wells using a bubble.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 17/242,098, filed Apr. 27, 2021, which is acontinuation application of U.S. patent application Ser. No. 16/272,970,filed Feb. 11, 2019, now U.S. Pat. No. 11,021,745, which is acontinuation of U.S. patent application Ser. No. 15/385,779, filed Dec.20, 2016, now U.S. Pat. No. 10,246,743, which is a continuation of U.S.patent application Ser. No. 14/521,425, filed Oct. 22, 2014, now U.S.Pat. No. 9,567,630, which claims the benefit of U.S. ProvisionalApplication No. 61/894,661, filed Oct. 23, 2013, each of which areentirely incorporated herein by reference.

BACKGROUND

Biochips can be used for various kinds of molecular detection andsensing, including the sequencing of nucleic acid molecules. Nucleicacid sequencing is a process that may be used to provide sequenceinformation for a nucleic acid sample. Such sequence information may behelpful in diagnosing and/or treating a subject. For example, thenucleic acid sequence of a subject may be used to identify, diagnose andpotentially develop treatments for genetic diseases. As another example,research into pathogens may lead to treatment for contagious diseases.

There are methods available which may be used to sequence a nucleicacid. Such methods, however, are expensive and may not provide sequenceinformation within a time period and at an accuracy that may benecessary to diagnose and/or treat a subject.

SUMMARY

Nanopores can be used to detect various molecules, including but notlimited to sequencing polymers such as nucleic acid molecules.Recognized herein is the need for improved biochips and methods formaking biochips (e.g., comprising nanopores). In some cases,conventional semiconductor processing techniques are deficient inproducing a silicon device for use as a biochip. For instance, methodsare provided that can produce a biochip that withstands (e.g., isoperable during or after contact with) highly corrosive environmentssuch as aqueous solutions, e.g., comprising ions. In another aspect, themethods described herein create a biochip surface conducive to theformation of organic membranes (e.g., lipid bilayers). In anotheraspect, the methods provide electrochemical electrodes needed to performelectrical measurements of ionic current flows in the biochip.

Amongst other things, the biochips produced according to the methodsdescribed herein can be used for nucleic acid molecule identificationand polymer (e.g., nucleic acid) sequencing. In some instances, thepolymer is passed through the nanopore and various subunits of thepolymer (e.g., adenine (A), cytosine (C), guanine (G), thymine (T)and/or uracil (U) bases of the nucleic acid) affect the current flowingthrough the nanopore. As described herein, the various subunits can beidentified by measuring the current at a plurality of voltages appliedacross the nanopore and/or membrane. In some cases, the polymerizationof tagged nucleotides releases and/or presents tag molecules to thenanopore that can be identified by measuring the current at a pluralityof voltages applied across the nanopore and/or membrane.

In an aspect, the disclosure provides a method for forming a lipidbilayer for use in a nanopore sensing device, comprising: (a) providinga chip comprising a fluid flow path in fluid communication with aplurality of sensing electrodes; (b) flowing a lipid solution into thefluid flow path; and (c) flowing at least one bubble onto the fluid flowpath, thereby forming a lipid bilayer adjacent to the sensingelectrodes, wherein the bubble spans the plurality of sensingelectrodes, and wherein the bubble is adjacent to the sensing electrodesfor at least about 1 second. In some embodiments, the bubble is adjacentto the sensing electrodes for between about 1 ms to about 5 minutes.

In some embodiments, the bubble is adjacent to the sensing electrodesfor at least about 30 seconds. In some embodiments, the bubble isadjacent to the sensing electrodes for at most about 5 minutes. In someembodiments, a lipid bilayer is formed over at least 50% of the sensingelectrodes. In some embodiments, a lipid bilayer is formed over at least70% of the sensing electrodes.

In some embodiments, the method further comprises inserting a nanoporeinto the lipid bilayers adjacent to each of the sensing electrodes. Insome embodiments, the chip comprises wells, and wherein the sensingelectrodes are in the wells.

In another aspect, the disclosure provides a method for forming a lipidbilayer for use in a nanopore sensing device, comprising: (a) providinga chip comprising a fluid flow path in fluid communication with aplurality of sensing electrodes; (b) flowing at least one bubble intothe fluid flow path and adjacent to said plurality of sensing electrodessuch that the bubble spans the plurality of sensing electrodes; and (c)contacting the periphery of the bubble with a lipid, wherein the lipiddiffuses under the bubble and onto the fluid flow path, thereby forminga lipid bilayer adjacent to the sensing electrodes.

In some embodiments, the bubble is contacted with the lipid for at leastabout 30 seconds. In some embodiments, the bubble is contacted with thelipid for between about 5 ms to about 5 minutes. In some embodiments, alipid bilayer is formed over at least 70% of the sensing electrodes. Insome embodiments, the method further comprises inserting a nanopore intothe lipid bilayers adjacent to each of the sensing electrodes. In someembodiments, the nanopore is Mycobacterium smegmatis porin A (MspA),alpha-hemolysin, any protein having at least 70% homology to at leastone of Mycobacterium smegmatis porin A (MspA) or alpha-hemolysin, or anycombination thereof.

In some embodiments, inserting the nanopore comprises applying anelectrical stimulus through said electrode to facilitate the insertionof said nanopore in said lipid bilayer. In some embodiments, said lipidbilayer exhibits a resistance greater than about 1 GΩ.

In some embodiments, said lipid bilayer and said nanopore proteintogether exhibit a resistance of about 1 GΩ or less. In someembodiments, said lipid comprises an organic solvent. In someembodiments, said bubble is a vapor bubble. In some embodiments, thechip comprises wells, and wherein the sensing electrodes are in thewells.

In some embodiments, said lipid is selected from the group consisting ofdiphytanoyl-phosphatidylcholine (DPhPC),1,2-diphytanoyl-sn-glycero-3phosphocholine,1,2-Di-O-Phytanyl-sn-Glycero-3-phosphocholine (DoPhPC),palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, sphingomyelin,1,2-di-O-phytanyl-sn-glycerol;1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl;GM1 Ganglioside, Lysophosphatidylcholine (LPC) or any combinationthereof.

In another aspect, the disclosure provides a nanopore sensing system,comprising: (a) a chip comprising a fluid flow path in fluidcommunication with a plurality of sensing electrodes, wherein each ofsaid sensing electrodes is configured to detect an ionic current upon anucleic acid incorporation event; and (b) a control system coupled tosaid chip, said control system programmed to: (i) flow a lipid solutioninto the fluid flow path; (ii) flow at least one bubble into the fluidflow path and adjacent to the sensing electrodes for a time period of atleast about 1 second, wherein the bubble spans the plurality of sensingelectrodes, and wherein the flow of the bubble into the fluid flow pathforms a lipid bilayer adjacent to the sensing electrodes. In someembodiments, the bubble is adjacent to the sensing electrodes for a timeperiod of between about 5 ms to about 5 minutes.

In some embodiments, the chip comprises wells, and wherein the sensingelectrodes are in the wells. In some embodiments, the control system isexternal to said chip. In some embodiments, the control system comprisesa computer processor. In some embodiments, the method further comprisesa fluid flow system operably coupled to said control system and saidchip, wherein said fluid flow system is configured to direct the flow ofsaid lipid solution and said bubble.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a pore-based electrosensor;

FIG. 2 shows a nanopore biochip;

FIG. 3 shows an electrode array where the container doubles as a counterelectrode;

FIG. 4A shows a top view of an electrode array with a common counterelectrode, and FIG. 4B shows a cross-section view of the electrode arraywith a common counter electrode of FIG. 4A;

FIG. 5 shows an electrode array where strips of sensors share a commoncounter electrode;

FIG. 6 shows and electrode array where each electrode has an independentcounter electrode;

FIG. 7 shows an example of rows of sensor wells sharing a commonelectrolyte pool;

FIG. 8 shows an example of a semiconductor substrate;

FIG. 9 shows a layer of silicon dioxide deposited on a semiconductorsubstrate;

FIG. 10 shows a photo-resist deposited on a silicon dioxide layer;

FIG. 11 shows an area of the photo-resist being exposed to radiation todefine the area of a well;

FIG. 12 shows a portion of the silicon dioxide being removed by a dryetch procedure;

FIG. 13 shows additional silicon dioxide being removed by a wet etchprocedure to create a well;

FIG. 14 shows deposition of a titanium adhesion layer;

FIG. 15 shows deposition of a titanium Nitride protective layer withPlatinum protective layer or alternately Platinum serving as theelectrode;

FIG. 16 shows deposition of silver electrode material;

FIG. 17 shows lift off of the photo-resist and materials disposedthereupon;

FIG. 18 shows silanization of the silicon dioxide;

FIG. 19 shows the filling of the well with a gel;

FIG. 20 shows creation of a membrane with a nanopore over the well;

FIG. 21 shows a biochip where the silver electrode comes up on the sidewalls of the well;

FIG. 22 shows a large bubble held adjacent to a plurality of electrodes;

FIG. 23 shows an example of a method for forming a lipid layer over theelectrodes on one or more flow channels of the primed sensor chip;

FIG. 24 shows an example of a semiconductor sensor chip;

FIG. 25A shows an example flowcell configuration wherein a gasket isplaced directly on top of a semiconductor chip having a rigid plastictop position on top of the gasket, FIG. 25B shows the flowcell of FIG.25A wherein the rigid plastic top is depicted transparently, and FIG.25C shows a top view of the flowcell of FIG. 25A wherein the rigidplastic top is depicted transparently;

FIG. 26 shows an example of a packaged chip; and

FIG. 27 shows an example of bilayer formation and pop automated with apump.

FIG. 28 is a flowchart for an automatic chip setup. This test wouldconfirm that the majority of cells on the chip are acceptable. If aninsufficient number of cells (as determined by the operator) pass thetest, then the entire chip will fail.

FIG. 29 is a flowchart for an automatic pump for bilayer formation.

FIG. 30 is an illustration of the flow of various solutions and/orbubbles over the wells of a sensor chip. The direction of flow isindicated by the block arrow in the lower right corner of the diagram.In this figure, the first rectangle representing ionic solution (3001;divot patterned rectangle) has already flowed over the wells (3010), thelipid solution (3015; cross-hatched rectangle) is on the chip (and inthis depiction covers all of the wells), and the second and thirdrectangles representing ionic solution, as well as the bubble (3005;clear rectangle) have not yet been flowed onto the chip. The size of therectangles is not representative of the amount of the fluid or size ofthe bubble. The ionic solution-bubble-ionic solution sequence may berepeated several times in order to increase the bilayer coverage,decrease the non-bilayer coverage, e.g., multi-layer stacks of lipids onthe wells, and/or reestablish the bilayer after a pop test. The lipidbilayer will form at the interface (3020) of the wells (shown) orsubstantially planar electrodes (not shown) once the method describedherein is performed.

The file of this patent contains at least one drawing in color. Copiesof this patent or patent publication with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “nanopore,” as used herein, generally refers to a pore, channelor passage formed or otherwise provided in a membrane. A membrane may bean organic membrane, such as a lipid bilayer, or a synthetic membrane,such as a membrane formed of a polymeric material. The membrane may be apolymeric material. The nanopore may be disposed adjacent or inproximity to a sensing circuit or an electrode coupled to a sensingcircuit, such as, for example, a complementary metal-oxide semiconductor(CMOS) or field effect transistor (FET) circuit. In some examples, ananopore has a characteristic width or diameter on the order of 0.1nanometers (nm) to about 1000 nm. Some nanopores are proteins. Alphahemolysin is an example of a protein nanopore.

The term “polymerase,” as used herein, generally refers to any enzymecapable of catalyzing a polymerization reaction. Examples of polymerasesinclude, without limitation, a nucleic acid polymerase or a ligase. Apolymerase can be a polymerization enzyme.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. A nucleotide caninclude A, C, G, T or U, or variants thereof. A nucleotide can includeany subunit that can be incorporated into a growing nucleic acid strand.Such subunit can be an A, C, G, T, or U, or any other subunit that isspecific to one or more complementary A, C, G, T or U, or complementaryto a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C,T or U, or variant thereof). A subunit can enable individual nucleicacid bases or groups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG,AC, CA, or uracil-counterparts thereof) to be resolved. In someexamples, a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), or derivatives thereof. A nucleic acid may besingle-stranded or double stranded.

A “polynucleotide” or “oligonucleotide” is a polymer or oligomercomprising one or more nucleotide as defined herein. A polynucleotide oroligonucleotide may comprise a DNA polynucleotide or oligonucleotide, aRNA polynucleotide or oligonucleotide, or one or more sections of DNApolynucleotide or oligonucleotide and/or RNA polynucleotide oroligonucleotide.

As used herein, a “nucleotide” or “base” can be a primary nucleotide ora nucleotide analog. A primary nucleotide is deoxyadenosinemono-phosphate (dAMP), deoxycytidine mono-phosphate (dCMP),deoxyguanosine mono-phosphate (dGMP), deoxythymidine mono-phosphate(dTMP), adenosine mono-phosphate (AMP), cytidine mono-phosphate (CMP),guanosine mono-phosphate (GMP) or uridine mono-phosphate (UMP). Anucleotide analog is an analog or mimic of a primary nucleotide havingmodification on the primary nucleobase (A, C, G, T and U), thedeoxyribose/ribose structure, the phosphate group of the primarynucleotide, or any combination thereof. For example, a nucleotide analogcan have a modified base, either naturally existing or man-made.Examples of modified bases include, without limitation, methylatednucleobases, modified purine bases (e.g., hypoxanthine, xanthine,7-methylguanine, isodG), modified pyrimidine bases (e.g.,5,6-dihydrouracil and 5-methylcytosine, isodC), universal bases (e.g.,3-nitropyrrole and 5-nitroindole), non-binding base mimics (e.g.,4-methylbezimidazole and 2,4-diflurotoluene or benzene), and no base(abasic nucleotide where the nucleotide analog does not have a base).Examples of nucleotide analogs having modified deoxyribose (e.g.,dideoxynucleosides such as dideoxyguanosine, dideoxyadenosine,dideoxythymidine, and dideoxycytidine) and/or phosphate structure(together referred to as the backbone structure) includes, withoutlimitation, glycol nucleotides, morpholinos, and locked nucleotides.

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between the nucleic acid sequence that encodes any oneof the inventive polypeptides or the inventive polypeptide's amino acidsequence, when aligned using a sequence alignment program.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence, e.g., the coding sequence for anyone of the inventive polypeptides, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet. See also, Altschul, et al., 1990 andAltschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402, 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 13.0.7, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

Biochips and Nucleic Acid Sequencing

Pore based sensors (e.g., biochips) can be used forelectro-interrogation of single molecules. A pore based sensor of thepresent disclosure can include a nanopore formed in a membrane that isdisposed adjacent or in proximity to a sensing electrode. The sensor caninclude a counter electrode. The membrane includes a trans side (i.e.,side facing the sensing electrode) and a cis side (i.e., side facing thecounter electrode).

Reference will now be made to the figures, wherein like numerals referto like parts throughout. It will be appreciated that the figures andfeatures therein are not necessarily drawn to scale.

With reference to FIG. 1 , a typical electrical measurement can operateon a molecule under test that is closely associated with a pore (e.g.,binding can be chemical, mechanical, electrical, or electrochemical).The system can apply a stimulus (voltage or current) across themolecule/pore complex and measure the response. In order to isolate themeasurement to the pore/molecule complex the two sides of the pore aregenerally separated by a highly insulating material (e.g., a lipidbilayer).

The volumes enclosed on the opposite sides of the insulating barrier arereferred to as the cis well and the trans well with the generaldefinition that the species of interest (e.g., the nucleic acid moleculeor tag molecule) moves from cis to trans during detection. The transwell is generally the side of the insulating membrane proximal to andelectrically connected to the chip electrodes.

FIG. 2 shows an example of a nanopore biochip (or sensor) havingtemperature control, as may be prepared according to methods describedin U.S. Patent Application Publication No. 2011/0193570, which isentirely incorporated herein by reference. With reference to FIG. 2 ,the nanopore detector comprises a top electrode 201 in contact with aconductive solution (e.g., salt solution) 207. A bottom conductiveelectrode 202 is near, adjacent, or in proximity to a nanopore 206,which is inserted in a membrane 205. The membrane 205 can be disposedover a well 210, or directly over an electrode, where the sensor 202forms part of the surface of the well. In some instances, the bottomconductive electrode 202 is embedded in a semiconductor 203 in which isembedded electrical circuitry in a semiconductor substrate 204. Asurface of the semiconductor 203 may be treated to be hydrophobic. Amolecule being detected goes through the pore in the nanopore 206. Thesemiconductor chip sensor is placed in package 208 and this, in turn, isin the vicinity of a temperature control element 209. The temperaturecontrol element 209 may be a thermoelectric heating and/or coolingdevice (e.g., Peltier device). Multiple nanopore detectors may form ananopore array.

In some embodiments, the biochip comprises a counter electrode capableof forming an electrical circuit with the electrode in the well. In somecases, the plurality of electrodes in the plurality of wells share acommon counter electrode. FIG. 3 shows an electrode array having acommon counter electrode where the liquid containment perimeter (e.g.,container) acts as a counter electrode (e.g., is conductive and forms acircuit). Another embodiment of a counter electrode is shown in FIG. 4A(top view) and FIG. 4B (cross-section view), where the counter electrodeis a plate (e.g., made of a conducting metal) over top of the nanopores.As shown in FIG. 5 and FIG. 6 , the plurality of electrodes in theplurality of wells can be organized into groups that share a commoncounter electrode. In some cases, (e.g., FIG. 6 ), the plurality ofelectrodes in the plurality of wells each have a dedicated counterelectrode. In some cases, having a plurality of counter electrodes canallow an individual sensing electrode, or only a few sensing electrodes,to be paired with a single counter electrode and thus potentiallyimprove the electrical response and performance of the sense-counterelectrode pairs.

In some cases, a plurality of wells (including any subset of the totalnumber of wells) comprise a common electrolyte pool. As shown in FIG. 7, the wells 701 may be separated into rows by walls 702 such that therow of wells share a common electrolyte pool above the wells. Separatingthe biochip into sections as described here can allow multiple samplesto be analyzed on a single biochip (e.g., by putting different samplesin different sections of the chip).

A nanopore sensor can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 100, or 1000 nanopores (e.g., hemolysin or aquaporin,etc. or combinations thereof) adjacent to a electrode (e.g., the bottomconductive electrode 202). A nanopore sensor can include a top electrode(e.g., the top electrode 201) that is for sole use by the nanoporesensor (and not other sensors), or as an alternative, a top electrodecan be provided for use by multiple nanopore sensors.

Biochip Processing

Controlling surface characteristics, well cavity volume, and electrodecomposition and volume can be major challenges of developing a scalablesemiconductor based planar array of microwells for the purpose ofnanopore sensing. In some instances, the ideal nanopore basedsemiconductor array sensing platform would achieve the following goals:(1) chip surface characteristics that support a planar insulatingmembrane, (2) differentiated surface characteristics that result in awell-defined and well controlled planar membrane surface, (3) largetrans-well electrolyte volume, (4) large electrode volume, (5) lowelectrical cross-talk between adjacent sensor electrodes on the array,(6) high cell density in order to achieve very large array sizes, and(7) stable measurements of very long duration during which the keyparameters (voltage, resistance, etc.) remain nearly constant.

For example meeting goals (1) & (2) can be difficult as in particular itcan be necessary to ensure that a highly insulating (resistive) barrieris formed with well controlled membrane areas and trans-well volumes.

In the case of forming a lipid bilayer membrane, the design andprocessing of the chip can be tailored to create hydrophobic (orlipophilic) surfaces and hydrophilic (or lipophobic) surfaces. Carefulcontrol of the chip surface allows well defined hydrophilic andhydrophobic areas to be defined. In turn this can control the structureand characteristics of the lipid bilayer membranes formed.

Goal (3) can be important in order to ensure that trans-wellelectrolytic ions are sufficiently abundant so as not to affect theresults during the duration of a typical measurement. This could occureither by depleting one or the other of the ions entirely or shiftingthe relative concentration of the various ions to such a degree thatthey change the measurement results substantially (i.e., through shiftsin concentration gradient and resulting Nernst potential).

Goal (4) can be important in the case of a sacrificial electrode that isconsumed or converted as part of the electro-chemical reaction thatsupports the measurement (e.g. silver being converted to silver-chlorideoxidation reaction). Having a high electrode volume can be importantboth to: (i) increase the time that a measurement can be continuouslyperformed without intervening “recharging” measurements which maydisrupt the experiment completely or result in gaps in the measured dataand (ii) reduce electrochemical potential shifts caused by the change inrelative concentrations of the oxidized and reduced electrodecomponents. In some cases, complete depletion of the electrode material(silver) sets a theoretical upper boundary on practical continuousmeasurement duration.

Unfortunately several of these goals can result in conflicts wheremeeting one goal comes at the expense of another. For example, etching adeep cavity in the silicon surface and filling completely with silvercan achieve a planar membrane at the metal/silicon surface, therebyachieving goals (1), (2), and (4) however leaves no remaining volumeavailable for trans-well electrolyte. Similarly, minimizing electricalcross-talk (goal 5) can be achieved by spacing adjacent cells far apart;however this comes at the expense of achieving goal (6).

In various aspects, the biochips and methods for making biochipsdescribed herein can achieve goals (1) to (6) in a way that is capableof sequencing nucleic acid molecules. For example, development of a deepwell vertical cavity structure to support both electrolyte and electrodematerial can meet goals (3) and (4); a hybrid wet/dry etch can increasethe lateral dimensions and thus trans well volume in can meet goals (1),(2), (3), and (4); selective silanization of oxide surfaces can achievegoals (1) and (2); utilization of a gel can be used to balance goals (3)and (4) while simultaneously achieving goals (1) and (2); implementationof distributed counter electrode can simultaneously achieve goals (5)and (6); use of electrode replenishment (recharging) can achieve goal(7); use of non-sacrificial electrodes (when feasible) can achieve goal(7); electro-plating can increase electrode material to meet goal (4);or any combination thereof.

Biochip Characteristics

In an aspect, a biochip comprises (a) a semiconductor substrate; (b) alayer of silicon dioxide disposed on the substrate, wherein a well isformed into the silicon dioxide; (c) a corrosion resistant materialcoating the inside of the well; (d) an electrode material in the wellfilling some fraction of that well including completely filling theoxide well to be coplanar with the surface of the oxide; and (e) anorganofunctional alkoxysilane layer coating the silicon dioxide. In someembodiments, the biochip further comprises a membrane isolating a firstfluid in the well from a second fluid outside the well. Also encompassedwithin the present invention are the biochips made by any of the methodsdescribed herein and the use of any of the biochips described herein orbiochips produced by the methods described herein to sequence polymers,including but not limited to nucleic acid molecules.

In some cases, electrode material is not depleted during operation ofthe biochip. In an aspect, a biochip comprises a plurality of wellshaving a membrane disposed over the well and an electrode in the wellthat is capable of detecting changes in the flow of ions through a porein the membrane in response to entities passing through the pore,wherein the electrode is not depleted during detection. In someembodiments, the electrode is substantially planar with the surface ofthe biochip, i.e., metal fills the entire well.

The electrode (e.g., silver or platinum material) can have any suitablemass or volume. In some cases, the volume of the electrode is about 0.1femto-liter (fL), about 0.5 fL, about 1 fL, about 5 fL, or about 10 fL.In some instances, the volume of the electrode is at least about 0.1femto-liter (fL), at least about 0.5 fL, at least about 1 fL, at leastabout 5 fL, or at least about 10 fL. In some embodiments, the volume ofthe electrode is at most about 0.1 femto-liter (fL), at most about 0.5fL, at most about 1 fL, at most about 5 fL, or at most about 10 fL.

The electrode can be made of any suitable material, including mixturesand alloys of materials. Some examples include platinum, silver, or anycombination thereof. In some cases, the electrode material is notconsumed during operation of the electrode. The electrode can comprise amaterial that has at least two oxidation states and/or a material thatis capable of both accepting and donating electrons.

Chip with Deep, Closely Packed Wells

Having a high density of nanopore sensors on the biochip may bedesirable for having a small device and/or sensing or sequencing a largenumber of molecules with a small biochip device. The surface comprisesany suitable density of discrete sites (e.g., a density suitable forsequencing a nucleic acid sample in a given amount of time or for agiven cost). In an embodiment, the surface has a density of discretesites greater than or equal to about 500 sites per 1 mm². In someembodiments, the surface has a density of discrete sites of about 100,about 200, about 300, about 400, about 500, about 600, about 700, about800, about 900, about 1000, about 2000, about 3000, about 4000, about5000, about 6000, about 7000, about 8000, about 9000, about 10000, about20000, about 40000, about 60000, about 80000, about 100000, or about500000 sites per 1 mm². In some embodiments, the surface has a densityof discrete sites of at least about 200, at least about 300, at leastabout 400, at least about 500, at least about 600, at least about 700,at least about 800, at least about 900, at least about 1000, at leastabout 2000, at least about 3000, at least about 4000, at least about5000, at least about 6000, at least about 7000, at least about 8000, atleast about 9000, at least about 10000, at least about 20000, at leastabout 40000, at least about 60000, at least about 80000, at least about100000, or at least about 500000 sites per 1 mm².

A biochip with a high density of discrete sites generally results in awell with a small area. In some instances, the well is suitably deep(e.g., such that the well has a suitably large volume). In someinstances, the well is substantially co-planar with the chip surface(i.e., metal fills the entire well). In an aspect, the volume of thewell is suitably large such that ion concentration is not fully depletedin the well before recharging the electrode. In an aspect, the electrodecan be a sacrificial electrode (e.g., an electrode that decreases and/orincreases in volume during detection, such as silver) and the volume ofthe well is suitably large such that the electrode is not fully depletedbefore recharging the electrode. In some embodiments, the well containsa sufficiently large volume of electrode material such as silver. Inthese aspects, amongst others, the volume of the well can limit the timefor which the electrode is capable of detecting a current (i.e., beforean ion is depleted and/or the electrode material is depleted).

In some embodiments, the wells have a suitably large volume such thatthe electrode can detect ion flow (e.g., current) for about 50 μs, about100 μs, about 150 μs, about 200 μs, about 250 μs, about 300 μs, about350 μs, about 400 μs, about 450 μs, about 500 μs, about 550 μs, about600 μs, about 650 μs, about 700 μs, about 750 μs, about 800 μs, about850 μs, about 900 μs, about 950 μs, about 1 ms, about 5 ms, about 10 ms,about 50 ms, about 100 ms, about 500 ms, about 1 s, about 5 s, about 10s, about 50 s, about 100 s, about 500 s, about 1000 s, or about 5000 s.In some embodiments, the wells have a suitably large volume such thatthe electrode can detect ion flow (e.g., current) for at least about 50μs, at least about 100 μs, at least about 150 μs, at least about 200 μs,at least about 250 μs, at least about 300 μs, at least about 350 μs, atleast about 400 μs, at least about 450 μs, at least about 500 μs, atleast about 550 μs, at least about 600 μs, at least about 650 μs, atleast about 700 μs, at least about 750 μs, at least about 800 μs, atleast about 850 μs, at least about 900 μs, at least about 950 μs, atleast about 1 ms, at least about 5 ms, at least about 10 ms, at leastabout 50 ms, at least about 100 ms, at least about 500 ms, at leastabout 1 s, at least about 5 s, at least about 10 s, at least about 50 s,at least about 100 s, at least about 500 s, at least about 1000 s, or atleast about 5000 s.

By balancing the potential voltage applied across the electrode andthereby recharging or redistributing the ions on either side of thebilayer pore, the data gathering lifetime of the pore may besignificantly extended to 10, 20, or 48 hours or longer. An examplewould be in nanopore system with 300 mM KCl ionic solution at pH7.5, toapply +120 mV across a bilayer pore for 30 seconds and then drop thevoltage to −120 mV for 40 seconds. The cycle is repeated in this slowswitching DC manner and the ionic charge distribution of the CIS andTRANS side of the bilayer pore remains balanced, as well as the thecomposition of Ag and AgCl present at one or more silver electrodes alsomaintains a balance. The result is a long life, data gathering poredetector. The level or magnitude of the positive and negatives voltagesand the time spent in + or − polarity can be varied to suit the salt orionic solution concentrations and the type of pore that is being used.

The time of detection can depend at least in part on the magnitude ofthe voltage applied across the nanopore and/or membrane (e.g., withhigher voltage magnitudes resulting in higher ion current, fasterdepletion of electrodes and therefore relatively shorter detectionperiods). In some embodiments, the voltage difference across themembrane is from about 0 mV to about 1V, positive or negative, e.g.,about 40 mV, about 60 mV, about 80 mV, about 100 mV, about 120 mV, about140 mV, about 160 mV, about 180 mV, about 200 mV, about 300 mV, about400 mV, or about 500 mV. In some embodiments, the voltage differenceacross the membrane is at most about 40 mV, at most about 60 mV, at mostabout 80 mV, at most about 100 mV, at most about 120 mV, at most about140 mV, at most about 160 mV, at most about 180 mV, at most about 200mV, at most about 300 mV, at most about 400 mV, or at most about 500 mV.In some embodiments, the voltage difference across the membrane is atleast about 0 mV to about 1V, positive or negative, e.g., at least about40 mV, at least about 60 mV, at least about 80 mV, at least about 100mV, at least about 120 mV, at least about 140 mV, at least about 160 mV,at least about 180 mV, at least about 200 mV, at least about 300 mV, atleast about 400 mV, or at least about 500 mV. The voltage can beconstant or variable (e.g., varying over any periodic waveform).

In some situations, the electrode has an operating life of at leastabout 1 minute (“min”), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min,9 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, or 12 hours under an appliedpotential of at least about 0 mV to about 1V, positive or negative,e.g., about 40 mV, about 60 mV, about 80 mV, about 100 mV, about 120 mV,about 140 mV, about 160 mV, about 180 mV, about 200 mV, about 300 mV,about 400 mV, or about 500 mV. In some examples, the electrode has anoperating life of at least about 15 min under an applied potential ofabout 80 mV.

The operating life of the electrode may be assessed based upon thedepletion (e.g., rate of depletion) of the electrode during use. In somecases, the electrode material is depleted by at most about 50%, 40%,30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or 0.01% withina time period that is less than or equal to about 60 minutes, 30minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3minutes, 2 minutes, or 1 minute during use of the electrode. In someembodiments, the electrode material is not depleted within a time periodthat is less than or equal to about 60 minutes, 30 minutes, 20 minutes,15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1minute during use of the electrode.

The wells can have any suitable depth. In some cases, the depth of thewell is measured from the surface of the biochip and/or bottom of themembrane to the top of the electrode and/or bottom of the electrode. Insome cases, the depth of the well is approximately equal to thethickness of an oxide layer (e.g., 203 in FIG. 2 ). In some embodiments,the wells are about 0.5 micrometers (μm), about 1 μm, about 1.5 μm,about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about4.5 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm,about 10 μm, or about 20 μm deep. In some embodiments, the wells are atleast about 0.5 micrometers (μm), at least about 1 μm, at least about1.5 μm, at least about 2 μm, at least about 2.5 μm, at least about 3 μm,at least about 3.5 μm, at least about 4 μm, at least about 4.5 μm, atleast about 5 μm, at least about 6 μm, at least about 7 μm, at leastabout 8 μm, at least about 9 μm, at least about 10 μm, or at least about20 μm deep.

In an aspect, a biochip comprises a plurality of wells having a membranedisposed over the well and an electrode in the well that is capable ofdetecting changes in the flow of ions through a pore in the membrane inresponse to entities passing through the pore. The biochip can compriseat least 500 wells per square millimeter and the wells can have asuitably large volume such that the electrode can detect at least 100entities without recharging the electrode.

In some embodiments, the entities are tag molecules detected duringnucleotide incorporation events. In some instances, a polymer passesthrough the pore and the entities are subunits of the polymer. In somecases, the polymer is a nucleic acid and the subunits of the polymer arenucleobases.

The biochip can detect any suitable number of entities withoutrecharging the electrode. In some cases, about 10, about 50, about 100,about 500, about 1000, about 5000, about 10000, about 50000, about100000, about 500000, about 1000000, about 5000000, or about 10000000entities are detected. In some cases, at least about 10, at least about50, at least about 100, at least about 500, at least about 1000, atleast about 5000, at least about 10000, at least about 50000, at leastabout 100000, at least about 500000, at least about 1000000, at leastabout 5000000, or at least about 10000000 entities are detected.

Chip with Closely Packed Wells and Minimum Cross-Talk

In an aspect, the wells are closely packed and have a low amount ofcross-talk (e.g., the electrodes derive all or most of their signal fromthe nanopore and/or membrane nearest to the electrode). In an aspect, abiochip comprises a plurality of wells having a membrane disposed overthe well and an electrode in the well that detects a signal in responseto the flow of ions, wherein the biochip comprises at least 500 wellsper square millimeter and the electrodes are electrically isolated fromeach other. The biochip can comprise any suitable number of wells perarea as described herein.

In some embodiments, an electrode detects about 80%, about 90%, about95%, about 99%, about 99.5%, or about 99.9% of its signal from the flowof ions through a nanopore in the membrane. In some instances, theelectrode detects at least about 80%, at least about 90%, at least about95%, at least about 99%, at least about 99.5%, or at least about 99.9%of its signal from the flow of ions through a nanopore in the membrane.In some cases, an electrode detects no more than 20%, no more than 10%,no more than 5%, no more than 1%, no more than 0.5%, or no more than0.1%, of its signal from the flow of ions through nanopores in adjacentwells.

Methods for Making Biochips

Certain methods can be used to make high quality biochips that are amongother things, capable of withstanding corrosive solutions and forming amembrane on the biochip that has a high resistivity. In an aspect, amethod for preparing a biochip comprises providing a semiconductorsubstrate and forming a plurality of wells containing electrodes capableof performing electrical measurements on or adjacent to the substratewhere the method further comprises (a) treating the substrate towithstand corrosive solutions; and (b) preparing the substrate for theformation of a membrane that seals the well with a high resistivity.

The membrane can have any suitably high resistivity. In some cases, theresistivity is about 10 mega-ohms (MΩ), about 50 MΩ, about 100 MΩ, about500 MΩ, about 1 giga-ohm (GΩ), about 5 GΩ, or about 10 GΩ. In somecases, the resistivity is at least about 10 mega-ohms (MΩ), at leastabout 50 MΩ, at least about 100 MΩ, at least about 500 MΩ, at leastabout 1 giga-ohm (GΩ), at least about 5 GΩ, or at least about 10 GΩ.

In some embodiments, the semiconductor substrate comprises silicon. Insome instances, the membrane is a lipid bilayer. The electrodes can becapable of measuring ionic current flows through a nanopore embedded inthe membrane.

The device can withstand any suitable corrosive solution. In some cases,the corrosive solutions are aqueous (include water) and comprise ions(e.g., Na+, Cl⁻). In some cases, the biochip is operable aftercontacting for many weeks with 1 M NaCl.

In an aspect, a method for preparing a biochip comprises: (a) depositinga material having reactive oxide groups on a semiconductor substrate;(b) etching wells into the silicon dioxide; (c) forming metal electrodesin the wells; (d) removing metal from all areas of the substrate exceptfor the wells; and (e) coating the substrate with a layer suitable foradhesion of a membrane. In some cases, the semiconductor substratecomprises silicon. The method can prepare the biochip for use in nucleicacid sequencing using a nanopore.

In some embodiments, the material in (a) is silicon dioxide. Thematerial can present a hard, planar surface that exhibits a uniformcovering of reactive oxide (—OH) groups to a solution in contact withits surface. These oxide groups can be the attachment points for thesubsequent silanization process (e). Alternatively, a lipophillic andhydrophobic surface material can be deposited that mimics the etchingcharacteristics of silicon oxide.

In some embodiments, a passivation layer is deposited on thesemiconductor substrate in (a), which may or may not have reactive oxidegroups. The passivation layer can comprise silicon nitride (Si3N4) orpolymide. In some instances, a photolithographic operation is used todefine regions where membranes form on the passivation layer.

FIG. 8 to FIG. 20 show an example of operations that can result inbiochips. All figures are not necessarily drawn to scale.

With reference to FIG. 8 , the method for producing a biochip can startwith a semiconductor substrate. The semiconductor (e.g., silicon) canhave any number of layers disposed upon it, including but not limited toa conducting layer such as a metal. The conducting layer is aluminum insome instances. In some cases, the substrate has a protective layer(e.g., titanium nitride). The layers can be deposited with the aid ofvarious deposition techniques, such as, for example, chemical vapordeposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD(PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hotwire CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axialdeposition (VAD), outside vapor deposition (OVD) and physical vapordeposition (e.g., sputter deposition, evaporative deposition).

In some cases, an oxide layer is deposited on the semiconductorsubstrate as shown in FIG. 9 . In some instances, the oxide layercomprises silicon dioxide. The silicon dioxide can be deposited usingtetraethyl orthosilicate (TEOS), high density plasma (HDP), or anycombination thereof.

In some instances, the silicon dioxide is deposited using a lowtemperature technique. In some cases, the process is low-temperaturechemical vapor deposition of silicon oxide. The temperature is generallysufficiently low such that pre-existing metal on the chip is notdamaged. The deposition temperature can be about 50° C., about 100° C.,about 150° C., about 200° C., about 250° C., about 300° C., about 350°C., and the like. In some embodiments, the deposition temperature isbelow about 50° C., below about 100° C., below about 150° C., belowabout 200° C., below about 250° C., below about 300° C., below about350° C., and the like. The deposition can be performed at any suitablepressure. In some instances, the deposition process uses RF plasmaenergy.

In some cases, the oxide is not deposited by a thermally grown oxideprocedure (e.g., which can use temperatures near or exceeding 1,000°C.).

The silicon dioxide can be deposited to a thickness suitable for theformation of wells comprising electrodes and a volume of electrolytecapable of sequencing at least 100, at least 1000, at least 10000, atleast 100000, or at least 1000000 nucleobases of a nucleic acid moleculewithout recharging the electrodes.

The silicon dioxide can be deposited to any suitable thickness. In someembodiments, the silicon dioxide is about 0.5 micrometers (μm), about 1μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm,about 4 μm, about 4.5 μm, about 5 μm, about 6 μm, about 7 μm, about 8μm, about 9 μm, about 10 μm, or about 20 μm thick. In some embodiments,the silicon dioxide is at least about 0.5 micrometers (μm), at leastabout 1 μm, at least about 1.5 μm, at least about 2 μm, at least about2.5 μm, at least about 3 μm, at least about 3.5 μm, at least about 4 μm,at least about 4.5 μm, at least about 5 μm, at least about 6 μm, atleast about 7 μm, at least about 8 μm, at least about 9 μm, at leastabout 10 μm, or at least about 20 μm thick.

Well Etching

Wells can be created in a silicon dioxide substrate using variousmanufacturing techniques. Such techniques may include semiconductorfabrication techniques. In some cases, the wells are created usingphotolithographic techniques such as those used in the semiconductorindustry. For example, a photo-resist (e.g., a material that changesproperties when exposed to electromagnetic radiation) can be coated ontothe silicon dioxide (e.g., by spin coating of a wafer) to any suitablethickness as shown in FIG. 10 . The substrate including the photo-resistis then exposed to an electromagnetic radiation source. A mask can beused to shield radiation from portions of the photo-resist in order todefine the area of the wells. The photo-resist can be a negative resistor a positive resist (e.g., the area of the well can be exposed toelectromagnetic radiation or the areas other than the well can beexposed to electromagnetic radiation as defined by the mask). In FIG. 11, the area overlying the location in which the wells are to be createdis exposed to electromagnetic radiation to define a pattern thatcorresponds to the location and distribution of the wells in the silicondioxide layer. The photoresist can be exposed to electromagneticradiation through a mask defining a pattern that corresponds to thewells. Next, the exposed portion of the photoresist is removed, such as,e.g., with the aid of a washing operation (e.g., 2% soln of TMAH (tetramethyl ammonium hydroxide) or other solution known to those of skill inthe art). The removed portion of the mask can then be exposed to achemical etchant to etch the substrate and transfer the pattern of wellsinto the silicon dioxide layer. The etchant can include an acid, suchas, for example, sulfuric acid (H₂SO₄). The silicon dioxide layer can beetched in an anisotropic fashion, though in some cases etching may beisotropic. For instance, with reference to FIG. 13 , an area notcorresponding exactly to the area of a final well can be etched (e.g.,the well can be etched under the photo-resist).

Various etching procedures can be used to etch the silicon dioxide inthe area where the well is to be formed. As shown in FIG. 12 and FIG. 13, the etch can be an isotropic etch (i.e., the etch rate alone onedirection is equal to the etch rate along an orthogonal direction), oran anisotropic etch (i.e., the etch rate along one direction is lessthan the etch rate alone an orthogonal direction), or variants thereof.

In some cases, an anisotropic etch removes the majority of the volume ofthe well. Any suitable percentage of the well volume can be removedincluding about 60%, about 70%, about 80%, about 90%, or about 95%. Insome cases, at least about 60%, at least about 70%, at least about 80%,at least about 90%, or at least about 95% of the material is removed inan anisotropic etch. In some cases, at most about 60%, at most about70%, at most about 80%, at most about 90%, or at most about 95% of thematerial is removed in an anisotropic etch. In some embodiments, theanisotropic etch does not remove silicon dioxide material all of the waydown to the semiconductor substrate. An isotropic etch removes thesilicon dioxide material all of the way down to the semiconductorsubstrate in some instances.

In some cases, the wells are etched using a photo-lithographic operationto define the wells followed by a hybrid dry-wet etch. Thephoto-lithographic operation can comprise coating the silicon dioxidewith a photo-resist and exposing the photo-resist to electromagneticradiation through a mask (or reticle) having a pattern that defines thewells. In some instances, the hybrid dry-wet etch comprises: (a) dryetching to remove the bulk of the silicon dioxide in the well regionsdefined in the photoresist by the photo-lithographic operation; (b)cleaning the biochip; and (c) wet etching to remove the remainingsilicon dioxide from the substrate in the well regions.

The biochip can be cleaned with the aid of a plasma etching chemistry,or exposure to an oxidizing agent, such as, for example, H2O2, O2, orO3. The cleaning can comprise removing residual polymer, removingmaterial that can block the wet etch, or a combination thereof. In someinstances, the cleaning is plasma cleaning. The cleaning operation canproceed for any suitable period of time (e.g., 15 to 20 seconds). In anexample, the cleaning can be performed for 20 seconds with an AppliedMaterials eMAx-CT machine with settings of 100 mT, 200 W, 20 G, 20 O2.

The dry etch can be an anisotropic etch that etches vertically (e.g.,toward the semiconductor substrate) but not laterally (e.g., parallel tothe semiconductor substrate). In some instances, the dry etch comprisesetching with a fluorine based etchant such as CF4, CHF3, C2F6, C3 F6, orany combination thereof. In one instance, the etching is performed for400 seconds with an Applied Materials eMax-CT machine having settings of100 mT, 1000 W, 20 G, and 50 CF4.

The wet etch can be an isotropic etch that removes material in alldirections. In some instances, the wet etch undercuts the photo-resist.Undercutting the photo-resist can make the photo-resist easier to removein a later operation (e.g., photo-resist “lift off”). In an embodiment,the wet etch is buffered oxide etch (BOE). In some cases, the wet oxideetches are performed at room temperature with a hydrofluoric acid basethat can be buffered (e.g., with ammonium fluoride) to slow down theetch rate. Etch rate can be dependent on the film being etched andspecific concentrations of HF and/or NH4F. The etch time needed tocompletely remove an oxide layer is typically determined empirically. Inone example, the etch is performed at 22° C. with 15:1 BOE (bufferedoxide etch).

The silicon dioxide layer can be etched to an underlying material layer.For example, with reference to FIG. 13 , the silicon dioxide layer isetched until the titanium nitride layer.

In an aspect, a method for preparing a biochip comprises etching wellsinto a silicon dioxide layer coated onto a semiconductor substrate using(a) a photo-lithographic operation to define the wells; (b) a dry etchto remove the bulk of the silicon dioxide in the well regions defined bythe photo-lithographic operation; and (c) a wet etch to remove theremaining silicon dioxide from the substrate in the well regions. Insome cases, the method further comprises removing residual polymer,removing material that can block the wet etch, or a combination thereof.The method can include a plasma cleaning operation.

As shown in FIG. 13 , the photo-resist is not removed from the silicondioxide following the photo-lithographic operation or the hybrid wet-dryetch in some cases. Leaving the photo-resist can be used to direct metalonly into the wells and not onto the upper surface of the silicondioxide in later operations. In some cases, the semiconductor substrateis coated with a metal (e.g., aluminum in FIG. 13 ) and the wet etchdoes not remove components that protect the metal from corrosion (e.g.,titanium nitride (TiN) in FIG. 13 ). In some cases, however, thephotoresist layer can be removed, with a wet chemistry such as SPM(sulfuric/peroxide mixture) or an organic solvent. In other embodiments,the photoresist layer may be removed with an oxygen plasma.

Electrode Metallization

Biochips described herein can be used to detect molecules and/orsequence nucleic acid molecules with aid of a nanopore and electricaldetection. Electrical detection can be performed with aid of anelectrode in the well and a counter-electrode located outside the well.Provided herein are methods for creating electrodes, such as metalelectrodes. The electrode can be reversibly consumed during detection,not consumed during detection, or not appreciably consumed duringdetection.

An example of an electrode that may be reversibly consumed duringmolecular detection is silver. An example of an electrode that may notbe appreciably consumed during detection is platinum.

An electrode can be formed adjacent to a substrate with the aid ofvarious deposition techniques. For instance, an electrode can be formedwith the aid of electroplating. As another example, an electrode can beformed with the aid of a vapor deposition techniques, such as, forexample, chemical vapor deposition (CVD), atomic layer deposition (ALD),plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organicCVD (MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD(MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD) andphysical vapor deposition (e.g., sputter deposition, evaporativedeposition).

In an aspect, a method for preparing a biochip comprises (a) providing asemiconductor substrate coated with a layer of silicon dioxide, where awell is etched into the silicon dioxide (e.g., as shown in FIG. 13 );(b) depositing a protective layer onto the well surface (e.g., TitaniumNitride or platinum as shown in FIG. 15 ); and (c) depositing theelectrode material onto the well surface (e.g., silver as shown in FIG.16 ). The method can further comprise depositing a film of adhesionmaterial onto the well surface to provide for adhesion and electricalconductivity of a metal layer to a layer below the metal layer. Theadhesion material can comprise titanium, tantalum, titanium nitride(TiN), chromium, or any combination thereof. With reference to FIG. 14 ,an adhesion material comprising titanium is deposited adjacent to thetitanium nitride layer, such as, for example, by electroplating, orvapor deposition (e.g., chemical vapor deposition). In some cases, asingle layer of metal replaces two or more layers (e.g., a single metallayer is both the adhesion layer and protective layer).

In some cases, the protective layer comprises a corrosive resistantmetal (e.g., platinum, gold). Without limitation, the protective layercan (i) provide electrical connectivity to an underlying conductor(e.g., to aluminum in FIG. 14 , or titanium nitride), (ii) protect theunderlying conductor from attack by a reactive solution (e.g., acorrosive solution such as sodium chloride in water), (iii) provide anelectron source and/or sink so that an electrode material is notconsumed in redox reactions (e.g., platinum can act as the source and/orsink when the electrode comprises silver), or (iv) any combinationthereof.

The various layers of metal (e.g., adhesion layer, protective layer,electrode material, etc.) can be deposited by any suitable technique,such as sputtering, deposition, electro-plating, or a combinationthereof. In some instances, the electrode material is deposited bysputtering, such as, for example, magnetron sputtering.

The electrodes are capable of making any suitable measurement asrequired for operation of the biochip. In some cases, the electrodematerial makes electrical measurements of analytes in the wells. Theanalytes can comprise nucleic acids, amino acids, proteins, tagmolecules, or any combination thereof. The electrical measurements canreversible redox reactions. In some embodiments, a sufficient volume ofthe electrode material is deposited into the well to provide fordetection of redox reactions involving analytes in the wells.

Lift-Off Procedure

There can be one or more layers of metal deposited onto the photo-resistfollowing electrode metallization as shown in FIG. 16 . In someinstances, the metal deposited onto the photo-resist is removed from thebiochip while the metal deposited in the wells remains in the wells.Leaving the photo-resist following creation of the wells (e.g., as shownin FIG. 13 ) can be advantageous for achieving metal removal from onlythe surface of the biochip and not the wells.

In some situations, following formation of a well and an electrode, thephotoresist may be omitted and metal outside of the electrode well canbe removed with the aid of a chemical mechanical polishing andsubsequent wet or reactive ion etching (RIE) etch if desired. In anexample, CMP is used to remove the electrode metal stack on the surfaceof the chip while it remains in the well (damascene process). In anotherexample, the photoresist and any overlying layer is removed usingacetone or another resist solvent (liftoff process).

Silanization of the Biochip Surface

Following formation of a well and electrode within the well, the silicondioxide layer can be treated to render the silicon dioxide layersuitable for forming a membrane in or adjacent to the well. In somecases, a hydrophobic membrane, such as, for example, a bilayer (e.g.,lipid bilayer), is formed over the well. The membrane can isolate theetched well from an overlying liquid, such as, for example, with aresistivity of at least about 10 gigaohms. As described herein,silanization of the silicon dioxide surface (e.g., to make the surfacehydrophobic) makes the surface suitable for formation of a membrane.

A method for stabilizing a membrane to a semiconductor interfacecomprises silanizing a semiconductor surface such that a membrane iscapable of adhering to the silanized surface and separating a firstfluid (e.g., on the cis side of the membrane) from a second fluid (e.g.,on the trans side of the membrane) with a resistivity of, for example,at least about 10 gigaohms.

A method for preparing a biochip can comprise: (a) providing a packagedbiochip or biochip precursor having a surface that comprises silicondioxide and/or metal (e.g., as shown in FIG. 17 ); and (b) silanizingthe surface (e.g., as shown in FIG. 18 ) using, for example, anorganofunctional alkoxysilane molecule. In some cases, theorganofunctional alkoxysilane molecule isdimethylchloro-octodecyl-silane, dimethylmethoxy-octodecyl-silane,methyldichloro-octodecyl-silane, trichloro-octodecyl-silane,trimethyl-octodecyl-silane, triethyl-octodecyl-silane or any combinationthereof.

The organofunctional alkoxysilane molecule can cover the silicon dioxidesurfaces (as shown in FIG. 18 ). The silane layer can be one molecule inthickness (FIG. 18 ).

Following silanization, the method can further comprise removingresidual silane from the substrate with a rinsing protocol. An examplerinsing protocol is a 5 second rinse with decane, acetone, ethanol,water, and ethanol followed by air drying and heating at 97° C. for 30minutes. The rinsing protocol can also used to clean the chip prior tothe application of the silane layer.

FIG. 21 shows that the silver and protective metal underneath cansputter onto the side walls of the wells and thus the silanization maynot come down into the well. In some instances, three fourths or more ofthe side walls of the wells are covered with silver and the protectivelayer underneath.

Formation of Bilayers

Described herein are methods for creating lipid bilayers and nanoporeson an array of electrodes (e.g., individually controlled) that make up asemiconductor nanopore sensor chip. The chip can be used for determininga polymer sequence, such as nucleic acid sequence, or the presence ofany tagged molecule.

Techniques for forming lipid bilayers over an array of electrodes on asemiconductor sensor chip are described herein. In an embodiment,liquids containing lipid molecules are inserted to the surface of thechip. The liquids can be separated by bubbles. The lipid molecules canbe distributed on the surface and the bubbles thin out the lipids tospontaneously form a lipid bilayer over each of the electrodes.Additional electrical stimulus may be applied to the electrodes tofacilitate the bilayer formation. Solutions containing nanopore proteinmay be further applied on top of the deposited lipids. More bubbles maybe rolled across the chip to facilitate the nanopore insertion into thebilayers. These techniques may occur with or without flow cells. In somecases, additional stimulus can be applied to induce bilayer or porecreation. Such stimulus can include pressure, sonication, and/or soundpulses. A stimulus may include any combination of buffers (pH range ofabout 5.0 to about 8.5), ionic solutions (e.g., NaCl, KCl; about 75 mMto about 1 M), bubbles, chemicals (e.g., hexane, decane, tridecane,etc.), physical movement, electrical stimulus or electrical stimuluspulses, pressure or pressure pulses, temperature or temperature pulses,sonication pulses, and or sound pulses to the sensor chip.

As shown in FIG. 22 , the bubble 2205 can be large and held adjacent toa plurality of wells 2210, each well 2210 containing an electrode. Thebubble can displace lipid from the region adjacent to the electrodes toproduce (a) lipid bilayer(s) that cover the electrode(s). In some cases,the edge of the bubble is contacted with a lipid solution 2215 and someof the lipid solution diffuses under 2220 the bubble 2205 to form (a)lipid bilayer(s) that cover the wells 2210 and the electrode(s). Thebubble can be a gas (or vapor) bubble. The bubble can include a singlegas or a combination of gases, such as, e.g., air, oxygen, nitrogen,argon, helium, hydrogen, or carbon dioxide. FIG. 30 also providesanother illustration of the bilayer formation methods as provided forherein.

The bubble can cover and/or be adjacent to any suitable number ofelectrodes. In some cases, the bubble is adjacent to about 100, about1000, about 10000, about 100000, about 1000000, or about 10000000electrodes. In some instances, the bubble is adjacent to at least about100, at least about 1000, at least about 10000, at least about 100000,at least about 1000000, or at least about 10000000 electrodes.

The bubble can remain adjacent to the electrodes for any suitable periodof time (e.g., long enough to form lipid bilayers). In some cases, thebubble is held adjacent to the electrodes for between about 10 ms toabout 10 minutes, e.g., 0.5 second (s), about 1 s, about 3 s, about 5 s,about 10 s, about 20 s, about 30 s, about 45 s, about 60 s, about 1.5minutes (min), about 2 min, about 3 min, about 4 min, about 5 min, orabout 10 min. In some cases, the bubble is held adjacent to theelectrodes for at least about 0.5 second (s), at least about 1 s, atleast about 3 s, at least about 5 s, at least about 10 s, at least about20 s, at least about 30 s, at least about 45 s, at least about 60 s, atleast about 1.5 minutes (min), at least about 2 min, at least about 3min, at least about 4 min, at least about 5 min, or at least about 10min. In some cases, the bubble is held adjacent to the electrodes for atmost about 0.5 second (s), at most about 1 s, at most about 3 s, at mostabout 5 s, at most about 10 s, at most about 20 s, at most about 30 s,at most about 45 s, at most about 60 s, at most about 1.5 minutes (min),at most about 2 min, at most about 3 min, at most about 4 min, at mostabout 5 min, or at most about 10 min. In some instances, the bubble isheld adjacent to the electrodes for between about 1 s and about 10 min,between about 10 s and about 5 min, or between about 30 s and about 3min.

In an aspect, a method for forming a lipid bilayer for use in a nanoporesensing device comprises providing a primed chip comprising a fluid flowpath in fluid communication with a plurality of sensing electrodes, andflowing a lipid solution into the fluid flow path and flowing a bubbleonto the fluid flow path, thereby forming a lipid bilayer adjacent toeach of the sensing electrodes. As used herein, a primed chip is a chipthat has had an initial flow of a KCl or an ionic solution over the chipand filling all the wells or channels. FIG. 7 shows an example of adevice having two fluid flow paths and FIG. 24 shows an example of adevice having 5 fluid flow paths. In some embodiments, the bubble spansthe plurality of sensing electrodes and is adjacent to the sensingelectrodes for at least about 1 second. The bubble can be adjacent tothe sensing electrodes or wells containing sensing electrodes for atleast between about 10 ms to about 10 minutes, e.g., 5 seconds, at leastabout 10 seconds, at least about 30 seconds and/or at most about 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes or 10 minutes. Insome cases, a nanopore is inserted into the lipid bilayers adjacent toeach of the sensing electrodes.

In an aspect, a method for forming a lipid bilayer for use in a nanoporesensing device comprises providing a primed chip comprising a fluid flowpath in fluid communication with a plurality of sensing electrodes,flowing a bubble onto the fluid flow path, where the bubble spans theplurality of sensing electrodes and contacting the periphery of thebubble with a lipid. The lipid can diffuse under the bubble and onto thefluid flow path (e.g., thereby forming a lipid bilayer adjacent to eachof the sensing electrodes). In some cases, the method further comprisesinserting a nanopore into the lipid bilayers adjacent to each of thesensing electrodes.

The bubble can be contacted with the lipid for any suitable period oftime (e.g., long enough to form lipid bilayers). In some cases, thebubble is contacted with the lipid for about 0.5 second (s), about 1 s,about 3 s, about 5 s, about 10 s, about 20 s, about 30 s, about 45 s,about 60 s, about 1.5 minutes (min), about 2 min, about 3 min, about 4min, about 5 min, or about 10 min. In some cases, the contacted with thelipid for at least about 0.5 second (s), at least about 1 s, at leastabout 3 s, at least about 5 s, at least about 10 s, at least about 20 s,at least about 30 s, at least about 45 s, at least about 60 s, at leastabout 1.5 minutes (min), at least about 2 min, at least about 3 min, atleast about 4 min, at least about 5 min, or at least about 10 min. Insome cases, the bubble is contacted with the lipid for at most about 0.5second (s), at most about 1 s, at most about 3 s, at most about 5 s, atmost about 10 s, at most about 20 s, at most about 30 s, at most about45 s, at most about 60 s, at most about 1.5 minutes (min), at most about2 min, at most about 3 min, at most about 4 min, at most about 5 min, orat most about 10 min. In some instances, the bubble is contacted withthe lipid for between about 1 s and about 10 min, between about 10 s andabout 5 min, or between about 30 s and about 3 min.

The method can form a lipid bilayer over any proportion of theelectrodes. In some cases, a lipid bilayer is formed over at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90% of the sensing electrodes. In some examples,a lipid bilayer is formed over about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or about100% of the sensing electrodes.

The bilayers can provide an electrical resistance between a solution onthe cis-side of the lipid bilayer and a solution on the trans-side ofthe bilayer. In some cases, the resistance is about 100 mega-ohm (MΩ),about 500 MΩ, about 1 giga-ohm (GΩ), about 10 GΩ, or about 100 GΩ. Insome cases, the resistance is at least about 100 mega-ohm (MΩ), at leastabout 500 MΩ, at least about 1 giga-ohm (GΩ), at least about 10 GΩ, orat least about 100 GΩ. In some embodiments, the resistance is about 1tera-ohm (TO).

Inserting the nanopore can comprise applying an electrical stimulus(e.g., voltage pulse or current pulse) through the electrode tofacilitate the insertion of the nanopore in the lipid bilayer. As analternative, or in addition to, a nanopore can be inserted by applyingone or more other stimuli, such as, for example, a pressure pulse, orany combination of buffers (pH range of about 5.0 to about 8.5), ionicsolutions (e.g., NaCl, KCl; about 75 mM to about 1 M), bubbles,chemicals (e.g., hexane, decane, tridecane, etc.), physical movement,electrical stimulus or electrical stimulus pulses, pressure or pressurepulses, temperature or temperature pulses, sonication pulses, and orsound pulses to the sensor chip. The nanopore can be any nanopore (e.g.,a protein nanopore). In some embodiments, the nanopore is Mycobacteriumsmegmatis porin A (MspA), alpha-hemolysin, any protein having at least70% homology to at least one of smegmatis porin A (MspA) oralpha-hemolysin, or any combination thereof.

In some instances, the resistance across the bilayer is reduced uponinsertion of a nanopore. The bilayers after nanopore insertion canprovide an electrical resistance between a solution on the cis-side ofthe lipid bilayer and a solution on the trans-side of the bilayer. Insome cases, the resistance after nanopore insertion is about 1 mega-ohm(MΩ), about 10 MΩ, about 100 MΩ, about 500 MΩ, about 1 giga-ohm (GΩ). Insome cases, the resistance after nanopore insertion is at most about 1mega-ohm (MΩ), at most about 10 MΩ, at most about 100 MΩ, at most about100 MΩ, at most about 500 MΩ, at most about 1 giga-ohm (Ge).

The lipid can be any suitable lipid or a mixture of lipids. In somecases, the lipid is dissolved in an organic solvent. In someembodiments, lipid is selected from the group consisting ofdiphytanoylphosphatidylcholine (DPhPC),1,2-diphytanoyl-sn-glycero-3-phosphocholine, Lysophosphatidylcholine(LPC), 1,2-Di-O-Phytanyl-sn-Glycero-3-phosphocholine (DoPhPC),palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol, sphingomyelin,1,2-di-O-phytanyl-sn-glycerol;1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl;GM1 Ganglioside, or any combination thereof.

In an aspect, a method for forming a lipid bilayer for use in a nanoporesensor, comprises directing a buffer solution in flow channel comprisingan electrode (or well containing an electrode) having a material layerthereon. The buffer solution can be electrically conductive, and thematerial layer can comprise one or more lipids. Next, the buffersolution can be brought in contact with the material layer, and one ormore voltages can be applied to the electrodes to encourage bilayerformation. Subsequently a current through the electrodes can be measuredto determine if at least a portion of the material layer has covered andsealed the electrodes and/or formed a bilayer over all or a portion ofthe electrode. The applied voltage may be sufficient to break thebilayer seal over the electrode and cause short circuit current flow.Based on a determination as to whether at least the portion of thematerial layer has covered and sealed the electrodes and/or formed abilayer over all or a portion of the electrode, a stimulus may beapplied simultaneously to all of the electrodes, groups of theelectrodes, or individual electrodes to induce at least the portion ofthe material layer to form the lipid bilayer adjacent to the electrode.

In some embodiments, the stimulus comprises at least one of a liquidflow over the surface of the electrode array, the sequential flow of oneor more different liquids over the surface of the array, the sequentialflow of any combination of one or more different liquids and bubblesover the surface of the array, an electrical pulse, sonication pulse,pressure pulse, or sound pulse. In some embodiments, the stimuluscomprises any combination of buffers (pH range of about 5.0 to about8.5), ionic solutions (e.g., NaCl, KCl; about 75 mM to about 1 M),bubbles, chemicals (e.g., hexane, decane, tridecane, etc.), physicalmovement, electrical stimulus or electrical stimulus pulses, pressure orpressure pulses, temperature or temperature pulses, sonication pulses,and or sound pulses. In some cases, the material layer comprises atleast two types of lipids. In some examples, the stimulus comprises thesequential flow of one or more liquids and bubbles over the surface ofthe array.

In an aspect, an automated method for creating a lipid bilayer on top ofeach one of multiple electrodes that make up an array of individuallycontrolled electrodes and a method to insert a single pore into eachbilayer atop each electrode in an array of individually controlledelectrodes on a semiconductor sensor is described. By applying anappropriate external stimulus (e.g., electrical stimulus, pressurestimulus, sonication, or sound) to a lipid layer in close proximity toan electrode on an essentially planar surface, a bilayer can be inducedto form over the electrode in an array of electrodes. Additionally, byapplying an appropriate external stimulus (e.g., including electricalstimulus, pressure stimulus, sonication, or sound) to an individualelectrode to the entire sensor chip that has lipid bilayers on one ormore electrodes and that are covered with a solution containing nanoporeproteins, a pore may be induced to insert into the bilayer. The resultis that a bilayer is created automatically, without manual intervention,over multiple electrodes in an array of individually controlledelectrodes in response to a stimulus and in a deterministic manner. Insome cases, a single nanopore can be inserted into multipleelectrode/bilayers in response to a stimulus and in a deterministicmanner and therefore create a highly parallel array of individuallycontrolled, electrical nanopore sensors. These arrays of individuallycontrolled nanopore sensors may be created on an essentially planarsemiconductor surface and that within the semiconductor material arecreated a portion or all of the circuitry needed to operate and controlthe individual electrodes.

In addition to the above approaches of creating bilayers and pores, thepresent disclosure provides methods to create bilayers and pores onarrays of individually controlled electrical/nanopore sensors that arecost effective and relatively simple and include 1) activating lipid orlipid-porin protein mixes already on the sensor (pre-applied) andcausing spontaneous bilayer creation or bilayer-pore creation, 2)activating lipid or lipid-porin protein mixes already on the sensor(pre-applied) and directly creating bilayers and or pores via electricalstimulation at the electrodes or stimulation to the system to createbilayers and or pores 3) activating lipid or lipid-porin protein mixesalready on the sensor (pre-applied) and directly creating bilayers andor pores via contacting a bubble to or running a bubble across thesurface of a sensor chip, 4) activating lipid or lipid-porin proteinmixes already on the sensor (pre-applied) and distributing, and thinningthe mixture on the surface of a sensor array using a bubble thatprepares the surface for subsequent electrical stimulation at theelectrodes or stimulation to the system to create bilayers and or pores,5) using a bubble to apply, distribute and thin a lipid mixture on thesurface of a sensor array so that bilayers are created over multipleindependent electrodes in an array, 6) using a bubble to apply,distribute and thin a lipid mixture and prepare the surface forsubsequent electrical stimulation at the electrodes or stimulation tothe system to create bilayers over multiple electrodes, 7) using abubble to apply, distribute and thin a porin protein mixture on thesurface of a sensor array prepared with a lipid mixture so that poresare inserted over multiple independent electrodes in an array, 8) usinga bubble to apply, distribute and thin a porin protein mixture andprepare the surface for subsequent electrical stimulation at theelectrodes or stimulation to the system to create a single pore overmultiple electrodes in an array, 9) using an electrical stimulus tocreate a bilayer over the surface of an electrode that does not requirethe generation or application of a bubble over the surface of anelectrode, 10) using sonication or pressure stimulus applied to one ormore electrodes, or to the entire sensor chip, to create a bilayerand/or pore over the surface of an electrode or multiple electrodes, 11)increasing the density of electrodes on a semiconductor array ofelectrodes for nanopore electrical sensing that is compatible with themethods for establishing bilayers and pores described above, 12) using asetup in which no flow cell or an open single sensor chip containing anarray of multiple electrode-nanopore sensors can support the methodsabove or elsewhere herein, or a single flow cell on a single sensor chipcontaining an array of multiple electrode-nanopore sensors can supportthe methods above or elsewhere herein, or multiple flow cells on asingle sensor chip containing an array of multiple electrode-nanoporesensors can support the methods above or elsewhere herein, 13) varying apressure of the liquid or bubble to improve successful bilayer or porecreation, and 14) varying a temperature of the senor chip and liquid toimprove bilayer or pore creation.

The present disclosure provides various approaches to create lipidbilayer and to insert a pore in the bilayer. In an embodiment, asemiconductor chip with multiple electrodes is presented. A liquid lipidsolution is applied to the silanized prepared surface of the chip. Theliquid lipid solution may be a solution of an organic solvent, e.g.,decane, hexane, tridecane, etc., and lipid molecules, such asdiphytanoylphosphatidylcholine (DPhPC) and/or any of the lipids notedabove. The solution may be applied on the surface by flowing, pouring,spraying, and/or squeegee. The solution is dried down on the surface.The solution may be substantially or completely dried so that onlypowder form of DPhPC molecules are left. As an alternative, the solutionmay be dried down to a sticky state. As such, the surface of the chipcan be functionalized by the pre-applied lipid molecules in a powderform or a sticky solution form. The chip is sealed and may be handledand shipped.

The semiconductor chip may contain a cover and the cover can allow theuser to pump in and pump out (or otherwise direct the movement of)liquid across the chip. In some examples, the user applies a bufferliquid (or solution), such as salt water, into the chip to activatelipid molecules, which may be in a dried or substantially dried state.Once the lipid molecules contact with the buffer solution, the lipidmolecules are hydrated. The pressure of the incoming buffer liquid mayfacilitate the formation of a lipid bilayer on top of each electrodesurface. The formation of the lipid bilayer may be spontaneous.

In some situations, the semiconductor chip may not contain a cover andthe user applies a buffer liquid (or solution), such as salt water, ontothe chip surface using a pipette or other fluid transfer and/or movementdevice (or instrument) to activate lipid molecules. Once the lipidmolecules contact with the buffer solution, the lipid molecules arehydrated. The pressure of the incoming buffer liquid may facilitate theformation of a lipid bilayer on top of each electrode surface.

In situations in which the semiconductor chip contains a cover, afterthe buffer liquid is applied into the chip, a bubble can be pumped in,and behind the bubble there is more buffer solution than in front of thebubble. The bubble sweeps across the chip and smoothes/thins out thenewly hydrated pre-deposited lipid mixture and causes the lipidmolecules to sweep across the surface. After the bubble flows through, alipid bilayer may be formed on top of each electrode surface.

In some cases, after the bubble is applied and sweeps across the chip,an electrical signal is applied to the electrode(s) and the electricalstimulus can cause bilayer(s) to form on the electrode(s). Theelectrical stimulus with a voltage potential can disrupt the interfacebetween the surface of the electrode and the lipid material around theelectrodes to cause the abrupt quick formation of bilayers.

In some embodiments, the liquid lipid solution may further contain poreproteins, such as Mycobacterium smegmatis porin A (MspA) oralpha-hemolysin. The solution containing lipid molecules and poreproteins are dried. The surface of the chip is prepared with silanemolecules to make the surface hydrophobic. Lipid molecules and poreproteins in are deposited in a powder form or in a sticky state. Theuser may activate the chip by applying a buffer solution to the chip.The lipid molecules and the pore proteins are hydrated. A lipid layerwith nanopore inserted may be formed on top of each electrode surface.The lipid layer may form spontaneously.

In some cases, after the buffer liquid is applied into the chip, abubble is pumped in. There may be more buffer solution behind the bubblethan in front of the bubble. The bubble can sweep across the chip andsmooth and thin out the newly hydrated pre-deposited lipid and pore mixand cause the lipid and/or pore molecules to sweep across the surface.After the bubble flows through, a lipid bilayer may be formed on top ofeach electrode surface in the manner described above or elsewhereherein, and pore proteins can be inserted in the bilayer to formnanopores.

As an alternative, or in addition to, after the bubble is applied andsweeps across the chip, an electrical signal can be applied to theelectrode and the electrical stimulus may cause a bilayer to form on theelectrode and nanopore to be inserted in the bilayer. The electricalstimulus with a voltage potential may disrupt the surface of theelectrode and affects the lipid material around the electrodes to causethe abrupt quick formation of bilayers and nanopores in the bilayers.

In another embodiment, the semiconductor chip is solely silanized anddoes not have any pre-applied molecules, such as lipid molecules or poreproteins, functionalizing the surface of the chip. The surface of thechip is initially flushed using salt water, i.e., primed. Then, analiquot of lipid in an organic solvent such as, for example, decane isinserted onto the chip. A bubble is followed to smear the lipid materialand distribute and thin out the lipid material on the surface of thechip. Lipid bilayers are created over multiple electrodes via contactand distribution of the bubble. The lipid bilayers may formspontaneously.

In another related embodiment, the lipid bilayers may not bespontaneously created after the bubble. A subsequent electricalstimulation and/or other stimuli is applied to the electrodes. It isbelieved that the electrical pulses and/or other stimuli assists increating a single bilayer by destroying multilayers and encouragingsingle bilayers to form over the electrode. The electrical pulse causesthe bilayers to be formed or destroyed on the electrodes.

In yet another related embodiment, KCL is flowed across the chip and thechip is wetted, i.e., primed. Then a small amount of lipid solution isapplied to the chip and flowed across the chip followed immediately by abubble that thins and distributes the lipid across the chip. Next saltwater is flowed across the chip. Following this a pore protein solutionis inserted into the chip. Another bubble is followed to smear and thinthe pore protein mixture on the surface of the chip so that pores areinserted over the multiple independent electrodes in an array via a formof contact or pressure from the bubble.

In still another related embodiment, after the pore protein solution andthe second bubble are inserted, a subsequent electrical stimulation isapplied at the electrodes to create nanopores in the lipid bilayers overthe multiple electrodes in an array.

In another embodiment, an aliquot of lipid in an organic solvent suchas, for example, decane gets inserted into the chip filled or coveredwith an ionic solution (such as salt water). A subsequent electricalstimulation is applied to the electrodes. The electrical pulse causesthe bilayers to be formed on the electrodes. In this embodiment, thereis no bubble inserted to facilitate bilayer formation. The lipid is welldistributed around the electrodes over the surface of the chip. Avoltage applied on the electrodes causes the disruption the lipidmaterial at the edge of the electrodes and induces formation of a lipidbilayer.

The semiconductor nanopore sensor chip may contain one or more channelsthrough which a liquid, solution and reagents can flow. In someembodiments, each channel has two rails, one on each side of thechannel. The electrodes may be on the bottom surface of the channel. Theelectrodes may further be on the sidewall surface of the channel (on therails). The density of electrodes for each channel may be increased bycreating electrodes on the bottom and sidewall surfaces.

One or more flow cells may be utilized on the semiconductor chip. Eachflow cell may be used to insert solutions and bubbles for one of thechannels on the chip. A flow cell is a path that liquids, bubbles andreagents can pass through. The channels on the chip acting as entire orportions of a flow cell may be independent so that the chip can processmultiple different samples independently and simultaneously.

In some embodiments, there is no channel or flow cell on the chip. Thechip is pre-applied with liquid lipid solution, or liquid lipid-poremixture solution. The solution can be dried to a powder form or a stickystate. A liquid buffer solution is applied to the chip to activate thelipid or lipid-pore mixture. An electrical signal is applied to theelectrode and the electrical stimulus may cause bilayer to form on theelectrode. The electrical stimulus with a voltage potential may disruptthe surface of the electrode and affects the lipid material around theelectrodes to cause the abrupt quick formation of bilayers. Furthermore,if there is activated pore protein present, the electrical stimulus mayfurther facilitate the insertion of pore molecules into the lipidbilayers.

In some embodiments, the pressure of the liquid or bubble may be variedto improve the bilayer or nanopore creation. In some embodiments, thetemperature of the chip and the liquid may be varied to improve thebilayer or pore creation. For example, slightly cooler than roomtemperature may be applied when the bilayer is formed; slightly warmerthan room temperature may be applied when the nanopore is inserted intothe lipid bilayer.

A chip may have one of the four sides of the sealed chip left open andaccessible. The opposite side may also have a single hole to which atube can contact and connect. If the chip is positioned or otherwisedisposed so that it is vertical with the hole and tube at the bottom andthe open end of the chip at the top, buffer liquid and reagents can beadded through the top and bubbles can then be released, at a controlledpace, from the bottom and travel up the sealed cavity and flow acrossthe chip. This system may not have trains of bubbles separating liquidfractions roll across the chip. It smoothes out any substances that areadded through the open top of the packaged chip and runs down thesurface of the chip inside. Conversely, it is possible to insert liquidsand reagents through the single tube at the bottom of the apparatus andthis may be advantageous when automated time series additions ofreagents may be required.

In some situations, sensor chips can be coupled to, or placed in, anapparatus that can automate the application of any combination ofliquids, reagents, bubbles, electrical stimulus pulses, pressure orpressure pulses, temperature or temperature pulses, sonication pulses,and or sound pulses to the sensor chip or liquid, reagent or bubble inthe sensor chip, to cause the automated creation of bilayers, creationof pores, maintenance of bilayers and pores including their re-creation,capture and reading of the biological molecules applied to the nanoporesensor chip, and to provide real-time and/or end-point details of thestatus of all sensors and all characteristics of the instrument'performance. The apparatus can allow any level of operator manualintervention or to allow creation of custom tests. The apparatus canapply different signals and/or reagents or act upon the sample or chipin response to the result of a prior test signal or reagent additionallowing the apparatus to operate fully or substantially automatically.Such a system can allow operator-free running of time-course experimentsor allow the refreshing of the nanopore system to re-functionalize thesurface of the sensor chip to continue testing.

The application of a stimulus to induce creation of bilayers or creationof pores can also include the application of any combination of buffers(pH range of about 5.0 to about 8.5), ionic solutions (e.g., NaCl, KCl;about 75 mM to about 1 M), bubbles, chemicals (e.g., hexane, decane,tridecane, etc.), physical movement, electrical stimulus or electricalstimulus pulses, pressure or pressure pulses, temperature or temperaturepulses, sonication pulses, and or sound pulses to the sensor chip tostimulate the desired or otherwise predetermined bilayer/pore creationevent(s).

A semiconductor chip may not contain a cover and the user may apply anyand all buffers, reagents, and bubbles manually through the use of apipette or other instrument. This manual application of these techniquescan be coupled with any applied stimulus outlined herein to induce thedesired bilayer and/or pore formation.

Flow cell or simple bubble systems of the present disclosure can alsogreatly help the insertion of pores by applying the pore proteinsolution evenly around the sensor chip surface and causing spontaneouspore insertion, or setting up the surface so that a stimulus of anycombination of buffers (pH range of about 5.0 to about 8.5), ionicsolutions (e.g., NaCl, KCl; about 75 mM to about 1 M), bubbles,chemicals (e.g., hexane, decane, tridecane, etc.), physical movement,electrical stimulus or electrical stimulus pulses, pressure or pressurepulses, temperature or temperature pulses, sonication pulses, and orsound pulses to the sensor chip can encourage the quick insertion ofpores into the bilayers. A flow cell or simple bubble system can alsohelp hydrate a dried lipid-pore-protein mix that may form bothspontaneous bilayers and pores after smoothing or mixing in anappropriate buffer with or without bubbles.

FIG. 23 illustrates a sample method for forming a lipid layer over theelectrodes on one or more flow channels of a primed sensor chip. Thesensor chip may be a planar chip that comprises multiple electrodesembedded in, and/or essentially planar to, a non-conductive orsemiconductor surface on which is located on the surface of flowchannels. The method comprises, in a first operation 2301, flowing in alipid solution comprising at least one type of lipid through each of theflow channels. Next, in a second operation 2302, the lipids aredeposited on the surface of and/or adjacent to electrodes. In a thirdoperation 2303, the deposited lipids are smoothed and thinned with afollow-on bubble in each of the flow channels. Next, in a fourthoperation 2304, each of the flow channels is once again filled with abuffer solution. The buffer solution can be electrically conductive. Ina fifth operation 2305, currents are measured through the electrodes todetermine if a lipid bilayer has been property formed over each theelectrodes. Next, in a sixth, optional, operation 2306, if the lipidbilayers have not been properly formed on any, all or substantially allof the electrodes, a stimulus (e.g., electrical stimulus) is applied toinduce the lipids on the surfaces to form lipid bilayers over theelectrodes. In some instances, however, the voltage is not applied tocreate bilayers.

In some embodiments, the lipid solution comprises at least two types oflipids. The lipid solution may further comprise at least one type ofpore proteins. The pore proteins may comprise Mycobacterium smegmatisporin A (MspA) or alpha-hemolysin. A non-lipid solution containing poreproteins can be directed over the deposited lipids in each of the flowchannels. The pore proteins and deposited lipids can then be thinnedwith a bubble in each of the flow channels. Next, a pore proteinsolution, an additional air (or gas) bubble and an additional liquidsolution can be directed through the flow channel. The pore proteinsolution and the liquid solution can be separated by the air bubble. Anelectrical stimulus can then be applied through at least some of theelectrodes to facilitate an insertion of the pore protein in the lipidbilayer. The operations of flowing solutions and bubbles may be repeatedin any order and combination to achieve the lipid bilayer formation andnanopore insertion in the bilayer. In some examples, the lipid arediphytanoylphosphatidylcholine (DPhPC),palmitoyl-oleoyl-phosphatidyl-choline (POPC),dioleoyl-phosphatidyl-methylester (DOPME), Lysophosphatidylcholine(LPC), 1,2-diphytanoyl-sn-glycero-3phosphocholine,1,2-Di-O-Phytanyl-sn-Glycero-3-phosphocholine (DoPhPC),dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidic acid,phosphatidylinositol, phosphatidylglycerol,1,2-di-O-phytanyl-sn-glycerol;1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl;GM1 Ganglioside, or sphingomyelin. The liquid lipid solution may furthercontain an organic solvent, such as decane.

In some embodiment, the buffer solution may contain ionic solution, suchas sodium chloride or potassium chloride solution. The buffer solutionmay further contain Ferrous Cyanide or Ascorbic Acid, sodium glutamate,potassium glutamate, tetramethylammonium chloride, tetraethylammoniumchloride, ammonium chloride, etc. Also may contain trehalose, sucrose,or any other sugar. The buffer may also contain divalents such asmagnesium chloride, calcium chloride, strontium chloride, manganesechloride, etc. In some embodiments, the pressure of the bubbles and/orfluid is adjusted substantially at or slightly above or below theatmospheric pressure to improve the bilayer formation or nanoporeinsertion.

FIG. 24 illustrates a sample semiconductor sensor chip, in accordancewith an embodiment of the present disclosure. The sensor chip 2400comprises multiple flow channels 2410. Each flow channel has multipleelectrodes 2440 embedded in, and planar or substantially planer to, anon-conductive or semiconductor surface on which is located on thesurface of the flow channels 2410. The surface of the flow channel otherthan the electrodes is hydrophobic. The surfaces of the flow channelother than the electrodes can be hydrophobic, hydrophilic, or anycombination thereof. Different walls may be treated for differentcharacteristics. The flow channels 2410 are separated by guide rails2420 along the flow channels. The channel width may be wide enough toaccommodate two or more rows electrodes. The electrodes may befabricated on the bottom surface of the flow channels, as well as theside walls of the guide rails, as shown in FIG. 24 . In someembodiments, the top side of the flow channels is sealed.

In another aspect, a method for forming a lipid bilayer over theelectrodes on one or more flow channels of a primed sensor chip, i.e., achip that has had the buffer solution flowed over the chip, comprises:(a) flowing in a lipid solution comprising at least one type of lipidsthrough each of the flow channels; (b) depositing the lipids on thesurface of the chip; (c) smoothing and thinning the deposited lipidswith a follow-on or additional bubble in each of the flow channels; (d)further flowing buffer solution through each of the flow channels, thebuffer solution being electrically conductive; (e) measuring currentsthrough the electrodes to determine if a lipid bilayer is formed overeach the electrodes; and (f) if the lipid bilayers are not formed on anyof the electrodes, optionally, applying a stimulus to at least one ofthe electrodes to induce the lipids on the surfaces to form lipidbilayers over the electrodes. The stimulus can comprise at least one ofan electrical pulse, sonication pulse, pressure pulse, and sound pulse,or any combination of buffers (pH range of about 5.0 to about 8.5),ionic solutions (e.g., NaCl, KCl; about 75 mM to about 1 M), bubbles,chemicals (e.g., hexane, decane, tridecane, etc.), physical movement,electrical stimulus or electrical stimulus pulses, pressure or pressurepulses, temperature or temperature pulses, sonication pulses, and orsound pulses to the sensor chip.

In some embodiments, the lipid solution comprises at least two types oflipids. In some embodiments, the lipid solution further comprises atleast one type of pore protein.

In some embodiments, after (c): a non-lipid solution containing poreproteins is directed over the deposited lipids in each of the flowchannels. The pore proteins and deposited lipids can then be thinnedwith a second bubble in each of the flow channels. The operations abovemay be repeated at least 1 time, 2 times, 3 times, 4 times, 5 times, ormore times in any order or combination.

In some cases, a pore protein solution, an additional air bubble and anadditional liquid solution can be directed through the flow channel. Thepore protein solution and the liquid solution can be separated by theair bubble. Next, a stimulus, for example, any combination of buffers(pH range of about 5.0 to about 8.5), ionic solutions (e.g., NaCl, KCl;about 75 mM to about 1 M), bubbles, chemicals (e.g., hexane, decane,tridecane, etc.), physical movement, electrical stimulus or electricalstimulus pulses, pressure or pressure pulses, temperature or temperaturepulses, sonication pulses, and or sound pulses to the sensor chip, canbe applied through at least some, all or substantially all of theelectrodes to facilitate an insertion of the pore protein in the lipidbilayer.

In some embodiments, the lipid is diphytanoylphosphatidylcholine(DPhPC), palmitoyl-oleoyl-phosphatidyl-choline (POPC),1,2-diphytanoyl-sn-glycero-3phosphocholine,1,2-Di-O-Phytanyl-sn-Glycero-3-phosphocholine (DoPhPC),dioleoyl-phosphatidyl-methylester (DOPME),dipalmitoylphosphatidylcholine (DPPC), Lysophosphatidylcholine (LPC),phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidic acid, phosphatidylinositol, phosphatidylglycerol,1,2-di-O-phytanyl-sn-glycerol;1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-350];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-550];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-750];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-1000];1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl;GM1 Ganglioside, or sphingomyelin.

In some cases, at least some of the liquid lipid solutions contain anorganic solvent (e.g., decane). The pore proteins, in some examples, cancomprise Mycobacterium smegmatis porin A (MspA) or alpha-hemolysin. Insome cases, the buffer solution contains an ionic solution containingone or more ions (e.g., sodium chloride or potassium chloride). In someinstances, at least some of the buffer solution contains ferrous cyanideor ascorbic acid. In some instances, the buffer solution may alsocontain sodium glutamate, potassium glutamate, tetramethylammoniumchloride, tetraethylammonium chloride, ammonium chloride, ferrocyanide,ferricyanide, potassium acetate, etc. In some instances, the buffersolution may also contain trehalose, sucrose, or any other sugar. Insome instances, the buffer solution may also contain divalents such asmagnesium chloride, calcium chloride, strontium chloride, manganesechloride, etc.

In some embodiments, the pressure of the bubbles and/or fluid issubstantially at or slightly above atmospheric pressure. The bubbles canhave a pressure that is greater than atmospheric, such as at a pressurethat is a magnitude of 101 kPa to 1013 kPa.

The surface of the metal electrodes is hydrophilic. In the instance whenthe electrodes may not be metal; such as conductive silicon, a potentialvoltage, or varying potential voltage, can be applied to the electrodesduring the silanization process to discourage silane from adhering andreacting to the non-metal electrodes. Voltages of ±up to ±2V anddiffering concentrations of low ionic buffer with the silane mix can beused. It is also possible to remove any reacted or residual silane frommetal or non-metal electrodes by cycling voltages at the electrodes and“burning off” the silane after deposition. For non-metal electrodes,after burning off a hydrophobic silane step a hydrophilic silane stepmay be added and only the space over the electrode will be open to reactto the silane. The result is an electrode surface that is nothydrophobic and not lipohillic and should be hydrophilic.

In some instances, the surface of the flow channel other than theelectrodes is hydrophobic. In some embodiments, the surfaces of the flowchannel (other than the electrodes) can be hydrophobic, hydrophilic, orany combination thereof. Different surfaces (walls or channel floor) maybe treated for different characteristics.

In some embodiments, before (a), the surface of the flow channel otherthan the electrodes can be rendered hydrophobic by silanizing,chemically treating, or using or designing specific materials, thesurface of the flow channel other than the electrodes; a plurality offlow channels can be formed on a surface of the chip; the electrodes canbe fabricated on a surface of each of the flow channels; the flowchannels can be separated by by building guide rails along the flowchannels; the electrodes can be fabricated on a side surface of each ofthe guide rails; and/or the top side of each of the flow channels can besealed.

In some situations, a chip having a bilayer can be created by flowing anionic solution across the chip. The flow can be a “train” ofinterspersed lipid solution and ionic solution aliquots (e.g.,alternating lipid solution and ionic solution). The flow can go throughsupply tubing and across the chip. In some examples, a train can have atleast or approximately 0.1 uL, 1 uL, 2 uL, 3 uL, 4 uL, or 5 uL of lipidand then at least or approximately 0.1 uL, 1 uL, 2 uL, 3 uL, 4 uL, or 5uL of ionic solution, and can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more times. The train of solutions can be pumped back and forthacross the surface of the biochip approximately 2, 3, 4, 5, 6, 7, 8, 9,10, or more times. The coverage and/or seal can then be electricallychecked. In some embodiments, the train of lipid and/or ionicsolution(s) may be between about 0.1 uL to about 1000 uL.

In some cases, the train of solutions is followed by an assemblyoperation. The assembly operation can involve flowing a bubble acrossthe chip. In some instances, electrical methods can be used to check thecoverage of cells (including electrodes) and/or leakage or sealresistance at each electrode.

In some cases, the assembly operation is repeated until at least some orall of the following test results are attained: (1) at least about 100,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or moreelectrodes are covered; (2) At least about 100, 110, 120, 130, 140, 150,160, 170, 180, 190, or 200 membranes (e.g., lipid layers) are popped atan applied voltage of less than −1V; (3) of the lipid layers that poppedin (2), at least 40, 50, 60, 80, 90, 100, or more have popped betweenabout −300 mV to −700 mV; (4) the number of electrodes with a sealresistance less than about 50 Giga-ohms is less than 30, 20, 15, or 10;and (5) if the number of cells which show any recorded leakage currentexceeds 50 then the median of the seal resistance is greater than 150Giga-ohms. In some cases, the assembly operation is repeated until atleast some or all of the following test results are attained: (1) atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more,e.g., 100%, electrodes are covered; (2) At least about 10%, 20%, 30%,40%, 50%, %, 70%, 80%, 90%, 95%, or more, e.g., 100%, membranes (e.g.,lipid layers) are popped at an applied voltage of less than ±1V; (3) ofthe lipid layers that popped in (2), at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more, e.g., 100%, have popped between about±300 mV to ±700 mV; and (4) a minimum number of cells that have greaterthan 10 GOhms (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%).

If some or all of these criteria are met, then a bubble of any sizebetween approximately 10 microliters (uL) to about 1000 uL can be flowedacross the chip, followed by an amount of buffer (e.g., about 10 uL toabout 10 mL) and a final test of (1), (4), and (5) can be performed. Ifthis passes, then the program moves to pore insertion protocol. Theprogram can be implemented with the aid of a computer system (e.g., uponexecution by a processor).

In some cases, the pore insertion protocol includes applying at least orabout uL, 1 uL, 2 uL, 3 uL, 4 uL, 5 uL or up to about 1 mL of poreprotein solution to the chip and applying a stimulus, e.g.,electroporating, to insert the pores into the bilayer. At the end of theelectroporation operation, the chip may be checked for pore yield and ifthe criteria are passed, sample and test reagents are applied.

The total time for bilayer creation and pore insertion can be anysuitable value. In some cases, the total time is about 1 minute, about 5minutes about 10 minutes, about 20 minutes, about 30 minutes, about 45minutes, about 1 hour, or about 2 hours. In some cases, the total timeis less than about 1 minute, less than about 5 minutes less than about10 minutes, less than about 20 minutes, less than about 30 minutes, lessthan about minutes, less than about 1 hour, or less than about 2 hours.In some instances, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, or about 90% of the total time isfor bilayer formation. Any proportion of the total time can be splitbetween bilayer formation and pore insertion. In some cases, the bilayeris formed and the nanopore is inserted simultaneously. In someinstances, the total time for bilayer and pore insertion is, on average,15 minutes for bilayer creation and 20 minutes for pore insertion for atotal of 35 minutes.

Any number of wells can be covered by a membrane (e.g., lipid bilayer)with inserted pore (e.g., pore yield). In some cases, the pore yield isabout 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, and the like. In some cases, the pore yieldis at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, and the like.

In some embodiments, the parameters applied to the electrode chip and toa test set-up are 1M KCl or 300 mM NaCl, pH 7.5 (pH range between about5.0 to about 8.5), current fluidic flow rates (e.g., between about 1uL/sec to about 1000 uL/sec), sea level atmospheric pressure, and roomtemperature.

Use of a Gel to Support the Membrane

In an aspect, a method for forming a biochip or sensor comprises coatinga substrate with a layer suitable for adhesion of a membrane (e.g., alipid bilayer comprising a nanopore). The substrate can be silanizedwith an organofunctional alkoxysilane molecule. FIG. 18 shows a biochipwhere a membrane can be disposed on the silanized surface.

In some cases, the membrane is difficult to form and/or is unstable atleast in part due to the membrane being supported on the silanizedsilicon dioxide, but not supported over the well. It is recognized anddescribed herein that filling the well with a gel can support themembrane over the well area, thereby making it easier to form themembrane and/or stabilizing the membrane. In some embodiments, the emptyportion of a well is filled with a gel as shown in FIG. 19 . The gel canprovide mechanical support for a membrane disposed over the well.

In other embodiments, the membrane may be stabilized chemically throughthe use of buffers comprising trehalose, or other sugars.

In an aspect, a method for preparing a biochip comprises: (a) depositinga gel into well that is in proximity to an electrode and sensingcircuit; and (b) forming a membrane over the well, wherein the membraneis at least partially supported by the gel.

In various embodiments, the gel is non-reactive, cross-linked, comprisesa liquid electrolyte, or any combination thereof. Gels can include butare not limited to standard reagent gels such as agarose andcommercially available proprietary gel matrixes. Examples are Collagen,Lamanin, Hydrogels, QGel, and HydroMax gels.

Insertion of a Nanopore

In some instances, a nanopore is inserted in the membrane (e.g., byelectroporation). The nanopore can be inserted by a stimulus signal suchas electrical stimulus, pressure stimulus, liquid flow stimulus, gasbubble stimulus, sonication, sound, vibration, or any combinationthereof. The nanopore can be a protein nanopore such as alpha-hemolysinor Mycobacterium smegmatis (MspA) nanopore or a nanopore that has atleast about 70% homology to either alpha-hemolysin or MspA.

In some embodiments, inserting the nanopore comprises applying astimulus (e.g., electroporation pulse) through said electrode tofacilitate the insertion of said nanopore. In some cases, this isfollowed by a second electrical detection pulse to detect the insertionof said nanopore in said lipid bilayer. The use of an electroporationpulse followed by detection pulse can be repeated quickly and/or manytimes and with sequentially varying voltage levels used for theelectroporation pulse until a pore is inserted and detection isachieved. In an embodiment, the initial electroporation pulse is about50 mV (positive or negative) one to ten times repeated with eachsubsequent batch of electroporation pulse(s) increasing from theprevious electroporation pulse by about 1 mV to a maximum of about ±700mV, i.e., a staircase of increasing voltage. The detection pulse is +160mV between each electroporation pulse. Thus, for example, the process ofinserting a nanopore in a lipid bilayer would be application of a 50 mVelectroporation pulse and application of a detection pulse five times,application of a 51 mV electroporation pulse and application of adetection pulse five times, etc. The process is repeated until ananopore is inserted in which case the electrode is turned off, or untilthe electrode/well is rejected or determined to have failed.

In some cases, an enzyme, e.g., polymerase (e.g., DNA polymerase) orother enzyme (e.g., reverse transcriptase), is attached to and/or islocated in proximity to the nanopore. The polymerase/enzyme can beattached to the nanopore before or after the nanopore is incorporatedinto the membrane. In some instances, the nanopore and polymerase/enzymeare a fusion protein (i.e., single polypeptide chain). It is to beunderstood that although a polymerase is exemplified throughout that anysuitable enzyme could be used.

The polymerase can be attached to the nanopore in any suitable way. Insome cases, the polymerase is attached to the hemolysin protein monomerand then the full nanopore heptamer is assembled (e.g., in a ratio ofone monomer with an attached polymerase to 6 hemolysin monomers withoutan attached polymerase). The nanopore heptamer can then be inserted intothe membrane.

Another method for attaching a polymerase to a nanopore involvesattaching a linker molecule to a hemolysin monomer or mutating ahemolysin monomer to have an attachment site and then assembling thefull nanopore heptamer (e.g., at a ratio of one monomer with linkerand/or attachment site to 6 hemolysin monomers with no linker and/orattachment site). It is understood that the combination of monomer witha linker and/or attachment site (H⁺) to hemolysin monomers with nolinker and/or attachment site (H⁻) may be done to achieve the heptamerichemolysin nanopore with any ratio of the subunits, e.g., (H⁺)₂(H⁻)₅,(H⁺)₃(H⁻)₄, (H⁺)₄(H⁻)₃, etc. A polymerase can then be attached to theattachment site or attachment linker (e.g., in bulk, before insertinginto the membrane). The polymerase can also be attached to theattachment site or attachment linker after the (e.g., heptamer) nanoporeis formed in the membrane. In some cases, a plurality ofnanopore-polymerase pairs are inserted into a plurality of membranes(e.g., disposed over the wells and/or electrodes) of the biochip. Insome instances, the attachment of the polymerase to the nanopore complexoccurs on the biochip above each electrode.

The polymerase can be attached to the nanopore with any suitablechemistry (e.g., covalent bond and/or linker). In some cases, thepolymerase is attached to the nanopore with molecular staples. In someinstances, molecular staples comprise three amino acid sequences(denoted linkers A, B and C). Linker A can extend from a hemolysinmonomer, Linker B can extend from the polymerase, and Linker C then canbind Linkers A and B (e.g., by wrapping around both Linkers A and B) andthus the polymerase to the nanopore. Linker C can also be constructed tobe part of Linker A or Linker B, thus reducing the number of linkermolecules. Linkers may also be biotin and streptavidin.

In some instances, the polymerase is linked to the nanopore usingSolulink™ chemistry. Solulink™ can be a reaction between HyNic(6-hydrazino-nicotinic acid, an aromatic hydrazine) and 4FB(4-formylbenzoate, an aromatic aldehyde). In some instances, thepolymerase is linked to the nanopore using Click chemistry (availablefrom LifeTechnologies for example). In some cases, zinc finger mutationsare introduced into the hemolysin molecule and then a molecule is used(e.g., a DNA intermediate molecule) to link the polymerase to the zincfinger sites on the hemolysin.

Methods for Detecting Bilayer Formation

After an attempt to create bilayers on the sensor described above, anelectrical stimulus can be applied to determine whether a bilayer hasbeen established or if the electrodes are simply covered with anon-bilayer layer. One way to do this is to apply a non-disruptive ACstimulus to the layer-covered electrodes and look for capacitive currentresponses that indicate the electrode is covered with a thin capacitivelipid bilayer (or other thin layer).

If appropriate capacitive readings are detected for the salt, voltage,and electrode diameter conditions then it can be inferred that a bilayerhas been created over the electrode and the operator is ready to beginthe pore insertion step.

Alternately, a distructive application of sequentially increasingvoltage pulses can be applied to each electrode of the array and thevoltage at which the layer over the electrode breaks is recorded. If thevoltages seen across an acceptable number of electrodes correspond toanticipated bilayer-break voltages for the salt, voltage, and electrodediameter conditions, then a single bubble is flowed across the chipre-make the bilayers and the operator is ready to begin the poreinsertion step.

Systems for Forming Wells and Nanopore Devices

Another aspect of the disclosure provides systems for forming nanoporedevices, including wells. Such systems can be used to form membranes(e.g., lipid bilayers) adjacent to the wells or electrodes, and insertnanopores in the membranes.

The system can include a deposition system, a pumping system in fluidcommunication with the deposition system, and a computer system (orcontroller) having a computer processor (also “processor” herein) forexecuting machine readable code implementing a method for forming thewells. The code may implement any of the methods provided herein. Thepumping system can be configured to purge or evacuate the depositionsystem. In some cases, the deposition system is precluded.

The deposition system can include one or more reaction spaces forforming material layers of the wells. In some situations, the depositionsystem is a roll-to-roll deposition system with one or moreinterconnected reaction chambers, which can be fluidically isolated fromone another (e.g., with the aid of purging or pumping at locationsin-between the chambers).

One or more deposition systems can be used to form a well. A depositionsystem can be configured for use with various types of depositiontechniques, such as, for example, chemical vapor deposition (CVD),atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasmaenhanced ALD (PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD),initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD),outside vapor deposition (OVD) and physical vapor deposition (e.g.,sputter deposition, evaporative deposition). A deposition system can beconfigured to enable layer-by-layer formation using varioussemiconductor manufacturing techniques, such as photolithography.

The pumping system can include one or more vacuum pumps, such as one ormore of a turbomolecular (“turbo”) pump, a diffusion pump, ion pump,cryogenic (“cryo”) pump, and a mechanical pump. A pump may include oneor more backing pumps. For example, a turbo pump may be backed by amechanical pump.

In some situations, an array comprising one or more wells is formed in asubstrate with the aid of a deposition system. Deposition may beregulated with the aid of a controller. In some embodiments, thecontroller is configured to regulate one or more processing parameters,such as the substrate temperature, precursor flow rates, growth rate,carrier gas flow rate and deposition chamber pressure. The controllerincludes a processor configured to aid in executing machine-executablecode that is configured to implement the methods provided herein. Themachine-executable code is stored on a physical storage medium, such asflash memory, a hard disk, or other physical storage medium configuredto store computer-executable code.

A controller can be coupled to various components of the system. Forinstance, the controller can be in communication with the one or moredeposition systems and/or fluid flow systems (e.g., pumping systems).The controller can be in communication with the pumping system, whichcan enable the controller to regulate a pressure of the enclosure.

A controller can be programmed or otherwise configured to regulate oneor more processing parameters, such as the substrate temperature,precursor flow rates, growth rate, carrier gas flow rate, precursor flowrate, and deposition chamber pressure. The controller, in some cases, isin communication with a valve or a plurality of valves of a depositionchamber, which aids in terminating (or regulating) the flow of aprecursor in the deposition chamber. The controller includes a processorconfigured to aid in executing machine-executable code that isconfigured to implement the methods provided herein. Themachine-executable code is stored on a physical storage medium, such asflash memory, a hard disk, or other physical storage medium configuredto store computer-executable code. The controller can also be used toregulate membrane and/or pore formation, such as the flow of a lipidsolution into a fluid flow path, the flow of one or more bubbles in thefluid flow path, and the application of one or more stimuli (e.g.,electrical stimulus).

Aspects of the systems and methods provided herein can be embodied inprogramming. Various aspects of the technology may be thought of as“products” or “articles of manufacture” typically in the form of machine(or processor) executable code and/or associated data that is carried onor embodied in a type of machine readable medium. Machine-executablecode can be stored on an electronic storage unit, such memory (e.g.,read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory ofthe computers, processors or the like, or associated modules thereof,such as various semiconductor memories, tape drives, disk drives and thelike, which may provide non-transitory storage at any time for thesoftware programming. All or portions of the software may at times becommunicated through the Internet or various other telecommunicationnetworks. Such communications, for example, may enable loading of thesoftware from one computer or processor into another, for example, froma management server or host computer into the computer platform of anapplication server. Thus, another type of media that may bear thesoftware elements includes optical, electrical and electromagneticwaves, such as used across physical interfaces between local devices,through wired and optical landline networks and over various air-links.The physical elements that carry such waves, such as wired or wirelesslinks, optical links or the like, also may be considered as mediabearing the software. As used herein, unless restricted tonon-transitory, tangible “storage” media, terms such as computer ormachine “readable medium” refer to any medium that participates inproviding instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

Methods for forming lipid bilayers, inserting nanopores in lipidbilayers, and sequencing nucleic acid molecules can be found in PCTPatent Publication No. WO2011/097028, which is incorporated herein byreference in its entirety. In some cases, the membrane is formed withaid of a bubble and the nanopore is inserted in the membrane with aid ofan electrical stimulus.

Described herein are uses of the biochips and/or biochips produced bythe methods described herein. The biochips may be used to determine thepresence of methylated nucleic acid bases in a sequence of nucleic acidbases.

The biochips described herein may be used to determine the effect ofdrugs or any man-made or naturally occurring molecule on the stabilityor performance of trans-membrane proteins or membrane bound proteins.The detector can be set up by creating an array (e.g., greater than 2)of individually addressable electrodes over which artificial or naturalcell membranes, or any insulating layer, are made as described herein.Into these membranes, layers, or insulating layers, any number ofpre-selected or unknown trans-membrane proteins may be inserted usingthe methods described herein. Any trans-membrane protein can be insertedinto the lipid bilayer (or any insulating layer) and the effects ofchemicals, drugs, and any biological or man-made molecule on thestability or performance of these trans-membrane proteins can beelectrically sensed and detected, for example by detecting thedisruption of the membrane after application of a specific drug. Anytrans-membrane protein whose presence can be detected ionically orelectrically provides even more information in the above assay aschanges in the molecules response to electrical stimulus can becorrelated with the application of specific drugs or changes in theenvironment impressed on the bilayer/pore.

The biochips described herein may be used to determine the effect ofdrugs or any man-made or natural molecules on the stability orperformance of different membranes placed over different portions of thearray sensor. By using the channels defined in the drawings of thisapplication different lipid bilayer materials or insulating layers maybe directed to different areas of the array chip, and a plurality ofdifferent lipid membranes or insulating layers can be presented to atest solution, each membrane/layer type present at a known location. Theability of drugs to influence membrane/layer types or any man-made ornaturally occurring molecule to effect the different membranes can bedetected.

The biochips described herein may be used to detect the presence of,capture, sort, and bin specific proteins or specific biomolecules in anunknown solution.

The biochips and methods of making and using biochips described hereincan use an electrolyte solution. In some cases, the ions in theelectrolyte solution flow through the nanopore and are detected by theelectrode. In cases where the electrode is a sacrificial electrode(i.e., depleted during detection, e.g., silver) the electrode can lastrelatively longer when the electrolyte comprises some salts rather thanothers. In some embodiments, the electrolyte does not comprise potassiumion (e.g., because potassium ion results in a relatively shorterelectrode life). In some embodiments, the electrolyte comprises lithiumchloride, tetramethylammonium chloride, triethylammonium chloride,ammonium chloride, sodium chloride, potassium glutamate, sodiumglutamate, or any combination thereof (e.g., because the listed saltsresult in a relatively shorter electrode life).

Biochips of the disclosure can perform sensing measurements with the aidof resistive, inductive or capacitive sensing. In some cases, a biochipcomprises an electrode that can sense a capacitance of a membraneadjacent to the electrode upon interaction of the membrane or a nanoporein the membrane with a species adjacent or in proximity to the membraneor the nanopore. Such measurements can be made with the aid of anapplied alternating current (AC) waveform or a direct current (DC)waveform.

EXAMPLES

The examples below are illustrative of various embodiments of thepresent disclosure and non-limiting.

Example 1. Forming Bilayers and Inserting Pores

Forming bilayers and inserting pores on the flow cell using a manualsyringe setup and an automated syringe pump setup results in highbilayer and single hemolysin pore yield. Bilayers are formed on bothsetups via flowing 1M or 0.3M KCl solution and air bubbles across alipid covered chip surface and applying electrical stimuli. Twohemolysin application methods result in high single pore yield. Onemethod involves the following operations: (1) premix hemolysin withlipid in decane, (2) flow the hemolysin-lipid mixture over the chipsurface and incubate for a few minutes, (3) form bilayers, and (4) applyan electrical stimulus to electroporate pores into bilayers. The secondmethod involves the following operations: (1) Flow KCl over the surfaceof the chip, (2) flow lipid in decane over the chip surface, (3) formbilayers, (4) flow hemolysin across the chip surface, or hemolysin andreaction mix across chip surface (5) apply an electrical stimulus toelectroporate pores into bilayers, and (6) flow KCl across chip surfaceto remove free hemolysin The method can be followed by reagent mixing orsimply leaving the hemolysin and reagent to mix on the chip beforebeginning to take readings. During the electroporation operation in bothapplication methods, the chip can be heated up to make bilayers morefluidic for easier hemolysin insertion. The temperature is reduced toroom temp or lower either during or after the electroporation operationto increase longevity of pore life.

Example 2. Flow Cell Configuration

With reference to FIGS. 25A-C-and FIG. 26 , the flow cell is assembledon the chip package by directly placing a gasket on top of thesemiconductor chip FIG. 25A. The gasket thickness varies from 50 um to500 um. The gasket can be composed of plastic with pressure sensitiveadhesives on one or both sides, silicone membrane, or flexibleelastomer, such as EPDM. The gasket can be made into any shape. A rigidplastic top (e.g., made from PMMA) is positioned on top of the gasket(e.g., made from PSA laminated PMMA) and can be sealed to the gasketthrough the pressure sensitive adhesive or by a locking mechanism thatapplies a compression force to the gasket. The top has single ormultiple inlet and outlet ports FIGS. 25B and 25C used to flow reagentsand air through the flow cell.

In some instances the overall gasket size is 4 mm by 4 mm square. Insome cases, the flowcell volume is about 1.5 ul for the 500 um thickgasket configuration. About to 20 electrodes are covered under thegasket in some embodiments.

Example 3. Bilayer Forming Protocol

-   -   1. Wet the chip surface by flowing over 300 mM KCl over        chip/through channels.    -   2. Flow through 20 uL 7.5 mg/ml DPhPC in decane followed by 120        uL 300 mM KCl, 20 mM HEPES, pH 7.5 (“KCl”).    -   3. Apply a series of negative electric pulses ranging from ±250        mV to ±1V with a 30 pA deactivation.    -   4. Wash chip with 2× (20 uL KCl, 20 uL bubble) then 120 uL KCl.    -   5. Repeat Step 3.    -   6. Repeat operations 4 and 5 until at least 30% of cells        deactivate between magnitude of 300 mV and 700 mV pulses (e.g.,        about 4 to 8 times).    -   7. Recover cells with 2× (20 uL KCl, 20 uL bubble) and 120 uL        KCl.

Step 6 is a destructive test to test for single lipid bilayers, versusmultilaminar, multistack or non-bilayer configurations. Optimalperformance is achieved with single bilayer configurations.

The bubbles used in steps 4 and 6, above, ranged from about 2 uL toabout 300 uL. The flow rate (of liquids and bubbles) ranged from about 1uL/sec to about 250 uL/sec, with a preferred flow rate of about 10uL/sec.

This was performed manually. An automated method is described in Example6, below.

Example 4. Pore Insertion Protocol

Method 1: mix hemolysin with lipid at start of experiment

-   -   1. After forming bilayers, set hand warmers on top of flow cell.    -   2. Electroporate pores into bilayers with a series of negative        electric pulses ranging from −50 mV to −600V with a 10 pA        deactivation.

The plate is then washed with 300 mM KCl to remove excess hemolysin.

Method 2: flow hemolysin over bilayers followed with a wash-firstelectroporation:

-   -   1. After forming bilayers, flow 20 ul of 100 ug/ml hemolysin in        0.3M KCl in 20 mM HEPES, pH 7.5 (“KCl”), and 5% glycerol through        flow cell.    -   2. Wash with 20 ul bubble and 80 uL 0.3M KCl, pH 7.5. Wash away        excess hemolysin with 300 mM KCl, pH 7.5.    -   3. Electroporate pores into bilayers with temperature set warmer        than room temperature.

Method 3: Bilayer Formation with hemolysin electroporation

-   -   1. Same as Method 2 except no wash step (Step 2).

Example 5. Bilayer Formation and Pop Automated with Pump

FIG. 27 shows the voltage at which the bilayer pops vs. cell locationunder repeated bilayer generation and wash conditions. Automated bubbleand KCl washing protocol allow consistent bilayer formation. Table 1shows bilayer formation and pop yield under various conditions (e.g.,with hemolysin and lipid or without hemolysin).

TABLE 1 Bilayer formation and pop Chip ID % Covered % Pop 120830_CC 01-1 99% 76% 120824_CC 06-1  94% 59% 120801_CC 01-1  92% 81% 120803_MT 01-1 73% 51% 120802_CC - 01-1  87% 93% 120731_MT 01-1 100% 89% 120803_MT01-1  73% 51% % Covered = number of cell that are covered by lipid atthe beginning; not necessarily a bilayer. % Pop = number of electrodesthat shorted the last time that the cells were popped.

Example 6: Fully Automated Bilayer Formation

This example provides a summary of the automated bilayer creationprotocol for a chip. This protocol can be separated into two sections:startup, verifying and preparing the chip for the bilayer formation, andthinning, the actual bilayer formation on the chip.

Startup

The startup protocol has three main steps: a dry check, a short checkand, optionally, conditioning. The dry check consists of applyingvoltages to each electrode to verify that none are giving anomalous datareadings. This is typically done by applying a voltage pulse andcounting cells that read current. If any actually give a current signal,then that electrode is deemed bad. If too many cells are bad, then theprocedure ends. The next step is a short check. The desired salt/buffersolution is flowed on the chip and a voltage is applied to eachelectrode to verify that all are giving a short circuit reading. This istypically done by applying a voltage pulse and counting cells that givea railed reading. If not enough cells are good (i.e., give the railedreading), then the procedure ends. The last step in the startup protocolis the optional conditioning. This step is used for faradaic electrodesand exercises the electrodes to get them into a state that is ideal forelectrochemistry. This is typically done by applying a series of voltagepulses and/or ramps to the cells.

Thinning

The thinning protocol has four main steps: the lipid addition, a leaktest, a bilayer pop and a bilayer recovery. Coming out of the startupprotocol, the chip is covered in the desired salt solution and theelectrodes are in a good state for the rest of the experiment. The firststep is to add lipid to the system. A small volume of the lipid/organicsolvent mixture is flowed over the chip, followed by more salt solution.We then enter a loop of events, that exits when the thinning protocolhas deemed the chip to have sufficient coverage and bilayer pops. Thefirst part of the loop is a leak test. This test applies a staircase ofincreasing voltage, and tests cells for two things: whether or not it iscovered by lipid material and if it is, what is the seal resistance ofthat coverage. If not enough cells are covered and have a high sealresistance, then a larger volume air bubble was flowed over the chip.This has been shown to increase our coverage across the chip. Anotherleak test followed this air bubble. This cycle will repeat untiladequate coverage and seal resistance is measured or too many tests failin a row, causing the procedure to end. Typically, it will pass afterthe first time and the procedure will move to the bilayer pop code. Thebilayer pop code applies square waves of increasing voltage, all the wayto ±1V. This is a destructive test that will pop bilayers, but coveredcells without a bilayer will not pop. Typically, not enough pop on thefirst round was observed, so a bubble was flowed over the chip. Not tobe bound by theory, it is believed that this bubble redistributes thelipid material, reforming bilayers over cells that popped, and alsothinning out the lipid material over cells that did not. After rounds ofleak tests, bilayer pops and bubbles, a threshold of popped cells willbe hit, indicating that bilayers have been formed over the bulk of thecells. After the chip is deemed to be complete, the chip is ready forthe insertion of nanopores.

Computer Implementation

The above protocol(s) may be automated and/or implemented on a computersystem. The computer-implemented method for producing a lipid bilayer onbiochip comprises:

-   -   a fluid dispensing device for selecting a fluid from a plurality        of fluid reservoirs and for dispensing each fluid onto a biochip        comprising a plurality of wells, wherein each fluid is dispensed        in a preselected order;    -   a plurality of fluid reservoirs, each reservoir containing a        fluid selected from a buffer, a lipid liquid, a wash liquid,    -   a bubble generating system, wherein bubbles are provided at        predetermined times;    -   an aspirator or gravity fed removal system for removing fluids        from the plurality of wells of the biochip;    -   a biochip comprising a plurality of electrodes, said electrodes        configured for detecting and/or determining a polymer sequence;        and        a control system for controlling the processing of the fluid and        bubble cycling to form the lipid bilayer on the biochip.

The bubble generating system may be an opening to the air such that abubble is created by pulling air from a valve port that is exposed toair, or by pulling gas from a valve port attached to a supply of a gas,and pushing it through the system.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-20. (canceled)
 21. A biochip, comprising: a substrate; and a plurality of discrete sites formed on the substrate, each discrete site comprising (a) a plurality of wells, the plurality of wells comprising a density greater than five hundred wells per square millimeter, (b) sidewalls disposed on the substrate to form each well, wherein the sidewalls have a planar top surface configured for a membrane to be formed thereon and wherein the membrane is configured to span across the well thereby sealing the well, and (c) an electrode disposed at the bottom of each well, wherein the electrodes disposed at the bottom of the wells are organized into a plurality of electrode groups and wherein each electrode group shares a common counter electrode.
 22. The biochip of claim 21, wherein the well is formed on the substrate by depositing on the substrate a material having reactive oxide groups and etching the well into the material.
 23. The biochip of claim 22, wherein the surfaces of the sidewalls are hydrophobic and wherein the hydrophobic surfaces facilitate formation of a hydrophobic membrane in or adjacent to the well.
 24. The biochip of claim 21, wherein the electrodes are electrically isolated from each other.
 25. The biochip of claim 24, wherein electrically isolating the electrodes from each other reduces cross-talk between wells.
 26. The biochip of claim 24, wherein electrically isolating the electrodes from each other reduces a signal from a nanopore or membrane formed atop a well with an electrode disposed at the bottom of a different well, thereby reducing cross-talk.
 27. The biochip of claim 21, wherein the electrode disposed at the bottom of the well derives signal from a nanopore or a membrane nearest to the electrode.
 28. The biochip of claim 21, wherein the electrode disposed at the bottom of the well has a dedicated counter electrode.
 29. The biochip of claim 21, wherein surfaces of the sidewalls are silanized.
 30. The biochip of claim 29, wherein the silanized surfaces facilitate formation of a membrane in or adjacent to the well.
 31. The biochip of claim 30, wherein facilitating the formation of a membrane in or adjacent to the well comprises facilitating adherence of the membrane to the silanized surfaces.
 32. The biochip of claim 29, wherein surfaces of the sidewalls are silanized by covering the sidewalls with a layer of organofunctional alkoxysilane molecules.
 33. The biochip of claim 32, wherein the layer of molecules is one molecule in thickness.
 34. The biochip of claim 21, wherein the substrate is a semiconductor substrate comprising a layer of silicon dioxide disposed on the substrate and wherein the well is formed into the silicon dioxide.
 35. The biochip of claim 21, wherein the membrane comprises a nanopore, and wherein the electrode is capable of detecting changes in the flow of ions through the nanopore in response to entities passing through the nanopore, wherein the wells have a suitably large volume such that the electrode can detect at least 100 entities without recharging the electrode, and wherein the electrode detects at least 80% of its signal from the flow of ions through the nanopore. 